SKBR3 Cells Research Articles

Anticancer and antiproliferative activities of Algerian Origanum majorana L.’s essential oil on PC-3 and SKBR3 cells

Anticancer and antiproliferative activities of Algerian <i>Origanum majorana</i> L.’s essential oil on PC-3 and SKBR3 cells

Differential Effect of 4H-Benzo[d] [1, 3]oxazines on the Proliferation of Breast Cancer Cell Lines.

A family of 4H-benzo[d][1,3]oxazines were obtained from a group of N-(2-alkynyl)aryl benzamides precursors via gold(I) catalysed chemoselective 6-exo-dig C-O cyclization. The precursors and oxazines obtained were studied in breast cancer cell lines MCF-7, CAMA-1, HCC1954 and SKBR-3 with differential biological activity showing various degrees of inhibition with a notable effect for those that had an aryl substituted at C-2 of the molecules. 4H-benzo[d][1,3]oxazines showed an IC50 rating from 0.30 to 157.4 µM in MCF-7, 0.16 to 139 in CAMA-1, 0.09 to 93.08 in SKBR-3, and 0.51 to 157.2 in HCC1954 cells. We observed that etoposide is similar to benzoxazines while taxol effect is more potent. Four cell lines responded to benzoxazines while SKBR-3 cell line responded to precursors and benzoxazines. Compounds 16, 24, 25 and 26 have the potent effect in cell proliferation inhibition in the 4 cell lines tested and correlated with oxidant activity suggesting a possible mechanism by ROS generation. These compounds represent possible drug candidates for the treatment of breast cancer. However, further trials are needed to elucidate its full effect on cellular and molecular features of cancer.

Expression Profiling Identified TRPM7 and HER2 as Potential Targets for the Combined Treatment of Cancer Cells.

TRPM7 is a divalent cation-permeable channel that is highly active in cancer cells. The pharmacological inhibitors of TRPM7 have been shown to suppress the proliferation of tumor cells, highlighting TRPM7 as a new anticancer drug target. However, the potential benefit of combining TRPM7 inhibitors with conventional anticancer therapies remains unexplored. Here, we used genome-wide transcriptome profiling of human leukemia HAP1 cells to examine cellular responses caused by the application of NS8593, the potent inhibitor of the TRPM7 channel, in comparison with two independent knockout mutations in the TRPM7 gene introduced by the CRISPR/Cas9 approach. This analysis revealed that TRPM7 regulates the expression levels of several transcripts, including HER2 (ERBB2). Consequently, we examined the TRPM7/HER2 axis in several non-hematopoietic cells to show that TRPM7 affects the expression of HER2 protein in a Zn2+-dependent fashion. Moreover, we found that co-administration of pharmacological inhibitors of HER2 and TRPM7 elicited a synergistic antiproliferative effect on HER2-overexpressing SKBR3 cells but not on HER2-deficient MDA-MB-231 breast cancer cells. Hence, our study proposes a new combinatorial strategy for treating HER2-positive breast cancer cells.

8028 Targeted Therapy-Induced ER And HER2 Chromatin Binding: A Novel Driver Of Poor Response To Treatment In Triple Positive Breast Cancer

Abstract Disclosure: S. Tam: None. M.D. McCoy: None. S. Bahnassy: None. R.B. Riggins: None. Triple positive breast cancer (TPBC) is a unique variant of breast cancer characterized by co-expression of two oncogenic drivers, estrogen receptor alpha (ER) and human epidermal growth factor receptor 2 (HER2). Both drivers promote tumor progression and serve as targets for effective small molecule and monoclonal antibody (mAb) therapies. Previous studies showed that the co-expression of ER reduces the efficacy of anti-HER2 targeted therapy in TPBC, and vice versa. ER is a well-established nuclear receptor and transcription factor. HER2 mainly functions as a transmembrane receptor, activating cytosolic pro-tumorigenic signaling pathways. However, less-studied nuclear HER2 is involved in the transcriptional regulation of several genes, leading to increased BC cell growth and survival. In this study, we investigate the subcellular localization and transcriptional regulatory functions of ER and HER2 in the unique context of TPBC, using cell fractionation, immunofluorescence, chromatin immunoprecipitation (ChIP)-qPCR, and integrated analysis of ER ChIP-seq/RNA-seq data. We hypothesize that crosstalk between ER and HER2 in the nucleus may, in part, underlie poor response to targeted therapy. At baseline, ER has strong nuclear localization in TPBC cells, BT474 and MDA-MB-361, but is predominantly cytosolic in ER+ MCF7 cells. Activated, phospho-HER2 is also localized to the nucleus of TPBC and HER2+ SkBr3 cells at baseline. Treatment with the anti-HER2 mAb combination of trastuzumab and pertuzumab (TP) induces ER binding to specific ER binding sites (ERBSs) in the enhancers or promoters of PGR, TFF1, and FOXC1 in BT474, but this is not observed in MCF7 or SkBr3 cells. Unlike TP, which increases HER2 nuclear localization but not ERBS recruitment, fulvestrant (a selective ER degrader) induces HER2 binding at the same ERBSs in BT474, but not MCF7 or SkBr3 cells. Collectively, these results suggest that the inhibition of either ER or HER2 can promote chromatin binding of the other at ERBSs. Integration of ER ChIP-seq peaks and differentially expressed genes from RNA-seq of BT474 cells treated with E2 vs control following short-term estrogen deprivation shows the ER cistrome is enriched for genes associated with RNA binding, RNA processing, and ribosome biogenesis. Interestingly, nuclear HER2 is reported to increase BC cell growth by activating transcription of ribosomal RNA genes. By contrast, the ER cistrome in MCF7 cells is enriched for genes associated with cell cycle regulation. In summary, anti-HER2 therapy induces recruitment of ER to well-established ERBSs, while anti-ER treatment causes recruitment of HER2 to these same sites in TPBC. Ongoing and future work leverages HER2 cistrome data to further define nuclear HER2/ER functional interactions, at the level of transcriptional regulation, that may contribute to poor treatment outcomes in TPBC. Presentation: 6/2/2024

8028 Targeted Therapy-Induced ER and HER2 Chromatin Binding: a Novel Driver of Poor Response to Treatment in Triple Positive Breast Cancer

Abstract Disclosure: S. Tam: None. M.D. McCoy: None. S. Bahnassy: None. R.B. Riggins: None. Triple positive breast cancer (TPBC) is a unique variant of breast cancer characterized by co-expression of two oncogenic drivers, estrogen receptor alpha (ER) and human epidermal growth factor receptor 2 (HER2). Both drivers promote tumor progression and serve as targets for effective small molecule and monoclonal antibody (mAb) therapies. Previous studies showed that the co-expression of ER reduces the efficacy of anti-HER2 targeted therapy in TPBC, and vice versa. ER is a well-established nuclear receptor and transcription factor. HER2 mainly functions as a transmembrane receptor, activating cytosolic pro-tumorigenic signaling pathways. However, less-studied nuclear HER2 is involved in the transcriptional regulation of several genes, leading to increased BC cell growth and survival. In this study, we investigate the subcellular localization and transcriptional regulatory functions of ER and HER2 in the unique context of TPBC, using cell fractionation, immunofluorescence, chromatin immunoprecipitation (ChIP)-qPCR, and integrated analysis of ER ChIP-seq/RNA-seq data. We hypothesize that crosstalk between ER and HER2 in the nucleus may, in part, underlie poor response to targeted therapy. At baseline, ER has strong nuclear localization in TPBC cells, BT474 and MDA-MB-361, but is predominantly cytosolic in ER+ MCF7 cells. Activated, phospho-HER2 is also localized to the nucleus of TPBC and HER2+ SkBr3 cells at baseline. Treatment with the anti-HER2 mAb combination of trastuzumab and pertuzumab (TP) induces ER binding to specific ER binding sites (ERBSs) in the enhancers or promoters of PGR, TFF1, and FOXC1 in BT474, but this is not observed in MCF7 or SkBr3 cells. Unlike TP, which increases HER2 nuclear localization but not ERBS recruitment, fulvestrant (a selective ER degrader) induces HER2 binding at the same ERBSs in BT474, but not MCF7 or SkBr3 cells. Collectively, these results suggest that the inhibition of either ER or HER2 can promote chromatin binding of the other at ERBSs. Integration of ER ChIP-seq peaks and differentially expressed genes from RNA-seq of BT474 cells treated with E2 vs control following short-term estrogen deprivation shows the ER cistrome is enriched for genes associated with RNA binding, RNA processing, and ribosome biogenesis. Interestingly, nuclear HER2 is reported to increase BC cell growth by activating transcription of ribosomal RNA genes. By contrast, the ER cistrome in MCF7 cells is enriched for genes associated with cell cycle regulation. In summary, anti-HER2 therapy induces recruitment of ER to well-established ERBSs, while anti-ER treatment causes recruitment of HER2 to these same sites in TPBC. Ongoing and future work leverages HER2 cistrome data to further define nuclear HER2/ER functional interactions, at the level of transcriptional regulation, that may contribute to poor treatment outcomes in TPBC. Presentation: 6/2/2024

Influence of PEGylation on HER2-targeting retro A9 peptide analogue

Elevated levels of HER2 receptor in breast cancer can be targeted through receptor-specific peptides for precise detection and therapy by nuclear medicine approach. Previously reported retro analogue of A9 peptide had shown HER2-specificity with promising pharmacokinetic features. Hence, with an aim of further improving the circulation time of rL-A9 radiopeptide, long polyethylene glycol chain (PEG12) was introduced at the N-terminus of the peptide during solid phase synthesis and influence of PEGylation on biological profile was studied. [177Lu]Lu-DOTA-PEG12-rL-A9 demonstrated high specific cellular uptake (5.94 ± 0.09 %) in HER2-expressing human breast carcinoma SKBR3 cells and low nanomolar binding affinity (Kd = 34.58 ± 12.78 nM). Uptake in SKBR3 tumors induced in female SCID mice was higher at all the time points investigated (3, 24, 48 h) in comparison to the non-PEGylated radiopeptide, [177Lu]Lu-DOTA-rL-A9. Blocking studies led to 51 % reduction in accumulation of radioactivity in the tumor indicating specificity of the radiopeptide. Improved tumor-to-stomach and tumor-to-intestine ratios for [177Lu]Lu-DOTA-PEG12-rL-A9 compared to [177Lu]Lu-DOTA-rL-A9 at 48 h shall pave the way for better contrast and delineation of metastatic sites.

Antitumor properties of bromelain loaded trastuzumab conjugated niosomes in HER2+ breast cancer cells

There are multiple forms of treatment for cancer treatment, but since the treatment process also damages healthy cells. Accordingly, in recent years, in the light of nanotechnological studies, cancer cell targeted drugs known as smart drugs have attracted attention. Niosome is one of the preferred nanoparticle systems in these studies. Bromelain is a protease mixture obtained from the pineapple stem or fruit. In recent years, it has been shown in many cell types that it has anticancer properties due to its proteolytic activity. In this study, bromelain was encapsulated into niosomes with 32 ± 2.9 % efficiency for the treatment of breast cancer, which is the most common type of cancer in women. Specific targeting of niosome-encapsulated bromelain to HER2+ SKBR-3 cell line was performed by trastuzumab (herceptin) conjugation. DLS, FTIR, XPS, TEM were used for particle characterization. Niosomes (Nio) showed particle size of 134.3 ± 8.1 nm, Bromelain loaded niosomes (Nio-Br) showed particle size of 165.6 ± 11.5 nm. As a result of TEM analysis, it was shown that morphologically spherical and targeted sizes of niosomes of approximately 100 nm were synthesized. FTIR and XPS analyzes were performed to confirm pegylation and trastuzumab conjugation. Cytotoxicity and apoptosis studies of Nio-Br, Nio-Br-PEG-Tra, Tra samples carried out in SKBR-3 and MDA-MB-231 cell lines. Determined IC50 values of final product on SKBR-3 and MDA-MB-231 cell lines respectively 77.51 ± 5.39 μg/mL and 238.1 ± 16.62 μg/mL. As a result of in vitro studies, final product is more toxic on HER2+ SKBR-3 cell line thanks to trastuzumab in structure. At the same time final product has more apoptotic effect connected to toxic characteristic. Formed molecule in this study was designed for HER2+ breast cancer cells. According to obtained in vitro experiment results, this designed molecule has potential to be drug delivery system for HER2+ breast cancer.

A Non-Canonical p75HER2 Signaling Pathway Underlying Trastuzumab Action and Resistance in Breast Cancer.

Overexpression of HER2 occurs in 25% of breast cancer. Targeting HER2 has proven to be an effective therapeutic strategy for HER2-positive breast cancer. While trastuzumab is the most commonly used HER2 targeting agent, which has significantly improved outcomes, the overall response rate is low. To develop novel therapies to boost trastuzumab efficacy, it is critical to identify the mechanisms underlying trastuzumab action and resistance. We recently showed that the inhibition of breast cancer cell growth by trastuzumab is not through the inhibition of HER2 canonical signaling. Here we report the identification of a novel non-canonical HER2 signaling pathway and its interference by trastuzumab. We showed that HER2 signaled through a non-canonical pathway, regulated intramembrane proteolysis (RIP). In this pathway, HER2 is first cleaved by metalloprotease ADAM10 to produce an extracellular domain (ECD) that is released and the p95HER2 that contains the transmembrane domain (TM) and intracellular domain (ICD). p95HER2, if further cleaved by an intramembrane protease, γ-secretase, produced a soluble ICD p75HER2 with nuclear localization signal (NLS). p75HER2 is phosphorylated and translocated to the nucleus. Nuclear p75HER2 promotes cell proliferation. Trastuzumab targets this non-canonical HER2 pathway via inhibition of the proteolytic cleavage of HER2 by both ADAM10 and γ-secretase. However, p75HER2 pathway also confers resistance to trastuzumab once aberrantly activated. Combination of trastuzumab with ADAM10 and γ-secretase inhibitors completely blocks p75HER2 production in both BT474 and SKBR3 cells. We concluded that HER2 signals through the RIP signaling pathway that promotes cell proliferation and is targeted by trastuzumab. The aberrant HER2 RIP signaling confers resistance to trastuzumab that could be overcome by the application of inhibitors to ADAM10 and γ-secretase.

Proteomic insights into breast cancer response to brain cell-secreted factors

The most devastating feature of cancer cells is their ability to metastasize to distant sites in the body. HER2 + and TN breast cancers frequently metastasize to the brain and stay potentially dormant for years until favorable conditions support their proliferation. The sheltered and delicate nature of the brain prevents, however, early disease detection and effective delivery of therapeutic drugs. Moreover, the challenges associated with the acquisition of brain biopsies add compounding difficulties to exploring the mechanistic aspects of tumor development. To provide insights into the determinants of cancer cell behavior at the brain metastatic site, this study was aimed at exploring the early response of HER2 + breast cancer cells (SKBR3) to factors present in the brain perivascular niche. The neural microenvironment was simulated by using the secretome of a set of brain cells that come first in contact with the cancer cells upon crossing the blood brain barrier, i.e., endothelial cells, astrocytes, and microglia. Cytokine microarrays were used to investigate the secretome mediators of intercellular communication, and proteomic technologies for assessing the changes in the behavior of cancer cells upon exposure to the brain cell-secreted factors. The cytokines detected in the brain secretomes were supportive of inflammatory conditions, while the SKBR3 cells secreted numerous cancer-promoting growth factors that were either absent or present in lower abundance in the brain cell cultures, indicating that upon exposure the SKBR3 cells may have been deprived of favorable conditions for optimal growth. Altogether, the results suggest that the exposure of SKBR3 cells to the brain cell-secreted factors altered their growth potential and drove them toward a state of quiescence, with broader overall outcomes that affected cellular metabolism, adhesion and immune response processes. The findings of this study underscore the key role played by the neural niche in shaping the behavior of metastasized cancer cells, provide insights into the cellular cross-talk that may lead cancer cells into dormancy, and highlight novel opportunities for the development of metastatic breast cancer therapeutic strategies.

177Lu-labeling of nuclear localization sequence (NLS)-grafted HER2-receptor affine peptide

Tagging of cell permeable nuclear localization sequence (NLS) with receptor targeting peptide vectors is an attractive strategy for selectively targeted translocation of therapeutic cargoes. The present study aimed at grafting nuclear localization sequence (NLS) onto breast cancer targeting rL-A9 peptide. Molecular docking analysis revealed higher binding affinity of the peptide, DOTA-NLS-rL-A9 (−26.1 kJ/mol) towards HER2 receptor in comparison to DOTA-rL-A9 peptide (−22.2 kJ/mol). Confocal microscopy data suggested significantly enhanced cellular internalization of NLS-tagged peptide. The engineered HER2-selective, DOTA-NLS-rL-A9 peptide scaffold was radiolabeled with Lu-177 for intracellular delivery of the theranostic radionuclide into tumor cells. [177Lu]Lu-DOTA-NLS-rL-A9 exhibited significantly enhanced binding affinity (4.58 ± 1.77 nM) towards human breast carcinoma SKBR3 cells and cellular internalization (85 % at 24 h) compared to its original analog, [177Lu]Lu-DOTA-rL-A9. In vivo biodistribution studies showed consistent retention of [177Lu]Lu-DOTA-NLS-rL-A9 in the tumor with negligible washout of radioactivity (∼4.1 % ID/g at 48 h). Prolonged tumor activity with rapid off-target tissue clearance resulted in significantly high tumor-to-background ratios. The radiopeptide, [177Lu]Lu-DOTA-NLS-rL-A9 thus, being precisely confined into HER2-expressing tumor cells and exhibiting favourable pharmacokinetic features is an efficient candidate for further screening.

RGS10 deficiency facilitates distant metastasis by inducing epithelial–mesenchymal transition in breast cancer

Distant metastasis is the major cause of death in patients with breast cancer. Epithelial–mesenchymal transition (EMT) contributes to breast cancer metastasis. Regulator of G protein-signaling (RGS) proteins modulates metastasis in various cancers. This study identified a novel role for RGS10 in EMT and metastasis in breast cancer. RGS10 protein levels were significantly lower in breast cancer tissues compared to normal breast tissues, and deficiency in RGS10 protein predicted a worse prognosis in patients with breast cancer. RGS10 protein levels were lower in the highly aggressive cell line MDA-MB-231 than in the poorly aggressive, less invasive cell lines MCF7 and SKBR3. Silencing RGS10 in SKBR3 cells enhanced EMT and caused SKBR3 cell migration and invasion. The ability of RGS10 to suppress EMT and metastasis in breast cancer was dependent on lipocalin-2 and MIR539-5p. These findings identify RGS10 as a tumor suppressor, prognostic biomarker, and potential therapeutic target for breast cancer.

RGS10 deficiency facilitates distant metastasis by inducing epithelial-mesenchymal transition in breast cancer.

Distant metastasis is the major cause of death in patients with breast cancer. Epithelial-mesenchymal transition (EMT) contributes to breast cancer metastasis. Regulator of G protein-signaling (RGS) proteins modulates metastasis in various cancers. This study identified a novel role for RGS10 in EMT and metastasis in breast cancer. RGS10 protein levels were significantly lower in breast cancer tissues compared to normal breast tissues, and deficiency in RGS10 protein predicted a worse prognosis in patients with breast cancer. RGS10 protein levels were lower in the highly aggressive cell line MDA-MB-231 than in the poorly aggressive, less invasive cell lines MCF7 and SKBR3. Silencing RGS10 in SKBR3 cells enhanced EMT and caused SKBR3 cell migration and invasion. The ability of RGS10 to suppress EMT and metastasis in breast cancer was dependent on lipocalin-2 and MIR539-5p. These findings identify RGS10 as a tumor suppressor, prognostic biomarker, and potential therapeutic target for breast cancer.

Targeted therapies in HER2-positive breast cancer with receptor-redirected Arazyme-linker-Herceptin as a novel fusion protein.

Targeted treatment of different types of cancers through highly expressed cancer cell surface receptors by fusion proteins is an efficient method for cancer therapy. The HER2 receptor is a member of the tyrosine kinase receptors family, which plays a notable role in breast cancer tumor development. About 25-30% of breast cancers overexpress human epidermal growth factor receptor 2 (HER2). In this study, we evaluated the particulars of a designed recombinant protein formed by HER2-specific Mab Herceptin linked with Arazyme on a HER2-overexpressing breast cancer cell line (SKBR3). Arazyme, a metalloprotease produced by Serratia proteamaculans was fused to the variable area of light and heavy chains of the Herceptin. The cytotoxic assay of the Arazyme-linker-Herceptin in the SKBR3 and MDA-MB-468 cells was evaluated by the MTT and flow cytometry techniques. The Caspase‑3 activity determination and adhesion assay were performed to evaluate the antitumor activity of the Arazyme-linker-Herceptin against SKBR3 cells. Furthermore, RT-PCR was used to measure the expression levels of the Bcl-2, Bax, MMP2, MMP9, and RIP3 genes. The Arazyme-linker-Herceptin showed higher cytotoxicity in SKBR3 cells compared to MDA-MB-468 cells. In addition, flow cytometry results revealed that the Arazyme-linker-Herceptin can significantly induce apoptosis in the HER2-overexpressing breast cancer cell line (SKBR3), which was confirmed by Bax upregulation and the decrease in adhesion of tumor cells and MMP2/MMP9. The findings of this study demonstrated that the Arazyme-linker-Herceptin induced apoptosis and decreased metastatic genes in SKBR3 cells; however, further research is required to confirm the effectiveness of the fusion protein.

CCDC88C, an O-GalNAc glycosylation substrate of GALNT6, drives breast cancer metastasis by promoting c-JUN-mediated CEMIP transcription

Coiled-coil domain containing 88C (CCDC88C) is a component of non-canonical Wnt signaling, and its dysregulation causes colorectal cancer metastasis. Dysregulated expression of CCDC88C was observed in lymph node metastatic tumor tissues of breast cancer. However, the role of CCDC88C in breast cancer metastasis remains unclear. To address this, the stable BT549 and SKBR3 cell lines with CCDC88C overexpression or knockdown were developed. Loss/gain-of-function experiments suggested that CCDC88C drove breast cancer cell motility in vitro and lung and liver metastasis in vivo. We found that CCDC88C led to c-JUN-induced transcription activation. Overlapping genes were identified from the genes modulated by CCDC88C and c-JUN. CEMIP, one of these overlapping genes, has been confirmed to confer breast cancer metastasis. We found that CCDC88C regulated CEMIP mRNA levels via c-JUN and it exerted pro-metastatic capabilities in a CEMIP-dependent manner. Moreover, we identified the CCDC88C as a substrate of polypeptide N-acetylgalactosaminyltransferase 6 (GALNT6). GALNT6 was positively correlated with CCDC88C protein abundance in the normal breast and breast cancer tissues, indicating that GALNT6 might be associated with expression patterns of CCDC88C in breast cancer. Our data demonstrated that GALNT6 maintained CCDC88C stability by promoting its O-linked glycosylation, and the modification was critical for the pro-metastatic potential of CCDC88C. CCDC88C also could mediate the pro-metastatic potential of GALNT6 in breast cancer. Collectively, our findings uncover that CCDC88C may increase the risk of breast cancer metastasis and elucidate the underlying molecular mechanisms.

Laser-assembled conductive 3D nanozyme film-based nitrocellulose sensor for real-time detection of H2O2 released from cancer cells

In this work, a nanostructured conductive film possessing nanozyme features was straightforwardly produced via laser-assembling and integrated into complete nitrocellulose sensors; the cellulosic substrate allows to host live cells, while the nanostructured film nanozyme activity ensures the enzyme-free real-time detection of hydrogen peroxide (H2O2) released by the sames. In detail, a highly exfoliated reduced graphene oxide 3D film decorated with naked platinum nanocubes was produced using a CO2-laser plotter via the simultaneous reduction and patterning of graphene oxide and platinum cations; the nanostructured film was integrated into a nitrocellulose substrate and the complete sensor was manufactured using an affordable semi-automatic printing approach. The linear range for the direct H2O2 determination was 0.5–80 μM (R2 = 0.9943), with a limit of detection of 0.2 μM. Live cell measurements were achieved by placing the sensor in the culture medium, ensuring their adhesion on the sensors' surface; two cell lines were used as non-tumorigenic (Vero cells) and tumorigenic (SKBR3 cells) models, respectively. Real-time detection of H2O2 released by cells upon stimulation with phorbol ester was carried out; the nitrocellulose sensor returned on-site and real-time quantitative information on the H2O2 released proving useful sensitivity and selectivity, allowing to distinguish tumorigenic cells. The proposed strategy allows low-cost in-series semi-automatic production of paper-based point-of-care devices using simple benchtop instrumentation, paving the way for the easy and affordable monitoring of the cytopathology state of cancer cells.

Ultrasonic-responsive piezoelectric stimulation enhances sonodynamic therapy for HER2-positive breast cancer

IntroductionBreast cancer ranks second as the most common malignancy globally, after lung cancer. Among the various subtypes of breast cancer, HER2 positive breast cancer (HER2 BC)poses a particularly challenging prognosis due to its heightened invasiveness and metastatic potential. The objective of this study was to construct a composite piezoelectric nanoparticle based on poly(vinylidene fluoride-trifluoroethylene) (P(VDF-TrFE)) for imaging and treatment of HER2 BC.MethodBy reshaping the crystal structure of P(VDF-TrFE) piezoelectric nanoparticles, improving hydrophilicity, and incorporating imaging capabilities, we developed piezoelectric composite nanoparticles (PGd@tNBs) that integrate imaging and therapeutic functions. The in vitro characterization encompassed the assessment of piezoelectric properties, hydrophilicity, imaging performance, and therapeutic efficacy of these particles. The targeting and therapeutic effectiveness of PGd@tNBs particles were further validated in the SK-BR3 cell line and subsequently confirmed in HER2-positive tumor-bearing mice.ResultsThe nanoparticle demonstrated excellent biocompatibility and impressive multimodal imaging performance. Magnetic resonance imaging (MRI) observations revealed significant accumulation of PGd@tNBs particles in the HER2 positive tumor, exhibiting superior contrast-enhanced ultrasound performance compared to traditional ultrasound contrast agents, and small animal in vivo imaging showed that PGd@tNBs particles were primarily excreted through respiration and urinary metabolism. Piezoforce Microscopy characterization highlighted the outstanding piezoelectric properties of PGd@tNBs particles. Upon targeted binding to HER2-BC, ultrasound stimulation influenced the cell membrane potential, leading to reversible electroporation. This, in turn, affected the balance of calcium ions inside and outside the cells and the mitochondrial membrane potential. Following ingestion by cells, PGd@tNBs, when exposed to ultrasound, triggered the generation of reactive oxygen species (ROS), resulting in the consumption of glutathione and superoxide dismutase and achieving sonodynamic therapy. Notably, repeated ultrasound stimulation, post PGd@tNBs particles binding and entry into cells, increased ROS production and elevated the apoptosis rate by approximately 45%.ConclusionIn conclusion, the PGd@tNBs particles developed exhibit outstanding imaging and therapeutic efficacy, holding potential for precise diagnosis and personalized treatment of HER2 BC.

Novel engineered HER2 specific recombinant protein nanocages for targeted drug delivery.

Protein nanocages resemble natural biomimetic carriers and can be engineered to act as targeted delivery systems, making them an attractive option for various drug delivery and biomedical applications. Our research investigated the genetic link of a specific anti-HER2 peptide (LTVSPWY) to the exposed N-terminal region of the maize (Zea mays) ferritin 1 (ZmFer1) protein nanocage, employing either a 7-amino acid (for LTVS-ZmFer1) or 16-amino acid (for LTVS-L-ZmFer1) linker. We utilized a heat treatment method to load the chemotherapeutic drug doxorubicin into the protein nanocage. The construct with the longer linker (LTVS-L) produced a greater amount of soluble protein nanocage and was selected for further experiments. The average size, polydispersity index, and zeta potential of the engineered protein nanocage were 19.01nm, 0.168, and - 2.13 mV, respectively. The LTVS-L-ZmFer1 protein nanocage exhibited excellent thermal stability, withstanding temperatures up to 100°C with only partial denaturation. Furthermore, we observed that cellular uptake of the LTVS-L-ZmFer1 protein nanocages in HER2-positive breast cancer cells was significantly higher compared to ZmFer1 after labeling with FITC (fluorescein isothiocyanate) (P-value = 0.0001). In addition, we observed a significant decrease in the viability of SKBR3 cells when treated with DOX-loaded LTVS-L-ZmFer1 protein nanocages compared to cells treated with DOX-loaded ZmFer1 protein nanocages. Therefore, this new treatment strategy may prove to be an effective way to reduce both the side effects and toxicity associated with conventional cancer treatments in patients with HER2-positive breast cancer.

Combined SERS-Raman screening of HER2-overexpressing or silenced breast cancer cell lines

BackgroundBreast cancer (BC) is a heterogeneous neoplasm characterized by several subtypes. One of the most aggressive with high metastasis rates presents overexpression of the human epidermal growth factor receptor 2 (HER2). A quantitative evaluation of HER2 levels is essential for a correct diagnosis, selection of the most appropriate therapeutic strategy and monitoring the response to therapy.ResultsIn this paper, we propose the synergistic use of SERS and Raman technologies for the identification of HER2 expressing cells and its accurate assessment. To this end, we selected SKBR3 and MDA-MB-468 breast cancer cell lines, which have the highest and lowest HER2 expression, respectively, and MCF10A, a non-tumorigenic cell line from normal breast epithelium for comparison. The combined approach provides a quantitative estimate of HER2 expression and visualization of its distribution on the membrane at single cell level, clearly identifying cancer cells. Moreover, it provides a more comprehensive picture of the investigated cells disclosing a metabolic signature represented by an elevated content of proteins and aromatic amino acids. We further support these data by silencing the HER2 gene in SKBR3 cells, using the RNA interference technology, generating stable clones further analysed with the same combined methodology. Significant changes in HER2 expression are detected at single cell level before and after HER2 silencing and the HER2 status correlates with variations of fatty acids and downstream signalling molecule contents in the context of the general metabolic rewiring occurring in cancer cells. Specifically, HER2 silencing does reduce the growth ability but not the lipid metabolism that, instead, increases, suggesting that higher fatty acids biosynthesis and metabolism can occur independently of the proliferating potential tied to HER2 overexpression.ConclusionsOur results clearly demonstrate the efficacy of the combined SERS and Raman approach to definitely pose a correct diagnosis, further supported by the data obtained by the HER2 gene silencing. Furthermore, they pave the way to a new approach to monitor the efficacy of pharmacologic treatments with the aim to tailor personalized therapies and optimize patients’ outcome.

Thermo and pH-responsive nanocarriers based on mesocellular silica foam and poly(N-vinylcaprolactam-co-methacrylic acid): Synthesis, characterization, and in vitro cytotoxicity assay

Smart hybrid nanocarriers were prepared by grafting poly(N-vinylcaprolactam-co-methacrylic acid) (poly(NVCL-co-MAA)) onto mesocellular silica foams (MCF silica) to evaluate them as controlled delivery systems of doxorubicin (DOX). The development of these nanocarriers began with the synthesis of MCF silica with interconnected porous structures and an average pore diameter of 27 nm. Then, free radical polymerization was used to graft the smart copolymer, and some parameters such as comonomer concentration and reaction time were studied. The DOX was loaded by immersion in an aqueous drug solution, while the in vitro release was investigated at different pHs (7.4, 5.8) and temperatures (25, 37 °C). In addition, the cytotoxicity of nanocarriers was investigated in vitro using the direct contact method with human breast cancer cells (SKBR3). In general, hybrid nanocarriers with copolymer graft percentages ranging from 30 to 60 % were obtained. On the other hand, at higher grafting of poly(NVCL-co-MAA) onto MCF silica, higher drug loading (up to 6.41 × 10−2 mg DOX/mg HN) was observed. Furthermore, in vitro DOX release results suggested that the nanocarriers released up to 52 % at 37 °C and a pH of 5.8. Finally, the in vitro cytotoxicity assay revealed that the DOX-loaded hybrid nanocarriers were cytotoxic to SKBR3 cells, indicating a controlled administration of DOX from the nanocarriers compared to free DOX. These results suggest that nanocarriers could have great potential for use as controlled delivery systems of anticancer drugs.

Study on the mechanism of heterogeneous tumor-associated macrophages in three subtypes of breast cancer through the integration of single-cell RNA sequencing and in vitro experiments.

Tumor-associated macrophages (TAM) exert a significant influence on the progression and heterogeneity of various subtypes of breast cancer (BRCA). However, the roles of heterogeneous TAM within BRCA subtypes remain unclear. Therefore, this study sought to elucidate the role of TAM across the following three BRCA subtypes: triple-negative breast cancer, luminal, and HER2. This investigation aimed to delineate the variations in marker genes, drug sensitivity, and cellular communication among TAM across the three BRCA subtypes. We identified specific ligand-receptor (L-R) pairs and downstream mechanisms regulated by VEGFA-VEGFR1, SPP1-CD44, and SPP1-ITGB1 L-R pairs. Experimental verification of these pairs was conducted by co-culturing macrophages with three subtypes of BRCA cells. Our findings reveal the heterogeneity of macrophages within the three BRCA subtypes, evidenced by variations in marker gene expression, composition, and functional characteristics. Notably, heterogeneous TAM were found to promote invasive migration and epithelial-mesenchymal transition (EMT) in MDA-MB-231, MCF-7, and SKBR3 cells, activating NF-κB pathway via P38 MAPK, TGF-β1, and AKT, respectively, through distinct VEGFA-VEGFR1, SPP1-CD44, and SPP1-ITGB1 L-R pairs. Inhibition of these specific L-R pairs effectively reversed EMT, migration, and invasion of each cancer cells. Furthermore, we observed a correlation between ligand gene expression and TAM sensitivity to anticancer drugs, suggesting a potential strategy for optimizing personalized treatment guidance. Our study highlights the capacity of heterogeneous TAM to modulate biological functions via distinct pathways mediated by specific L-R pairs within diverse BRCA subtypes. This study might provide insights into precision immunotherapy of different subtypes of BRCA.