scholarly journals PE38-based gene therapy of HER2-positive breast cancer stem cells via VHH-redirected polyamidoamine dendrimers

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Cobra Moradian ◽  
Fatemeh Rahbarizadeh
Keyword(s):  
Breast Cancer ◽  
Stem Cells ◽  
Gene Therapy ◽  
Growth Factor ◽  
Cancer Stem Cells ◽  
Toxin Gene ◽  
Breast Cancer Stem Cells ◽  
Hanging Drop ◽  
Her2 Positive ◽  
Mammosphere Formation

AbstractBreast cancer stem cells (BCSCs) resist conventional treatments and cause tumor recurrence. Almost 25% of breast cancers overexpress human epidermal growth factor receptor-2 (HER2). Here we developed a novel multi-targeted nanosystem to specifically eradicate HER2+ BCSCs. Plasmids containing CXCR1 promoter, PE38 toxin, and 5′UTR of the basic fibroblast growth factor-2 (bFGF 5'UTR) were constructed. Polyamidoamine (PAMAM) dendrimers functionalized with anti-HER2 VHHs were used for plasmid delivery. Stem cell proportion of MDA-MB-231, MDA-MB-231/HER2+ and MCF-10A were evaluated by mammosphere formation assay. Hanging drop technique was used to produce spheroids. The uptake, gene expression, and killing efficacy of the multi-targeted nanosystem were evaluated in both monolayer and spheroid culture. MDA-MB-231/HER2+ had higher ability to form mammosphere compared to MCF-10A. Our multi-targeted nanosystem efficiently inhibited the mammosphere formation of MDA-MB-231 and MDA-MB-231/HER2+ cells, while it was unable to prevent the mammosphere formation of MCF-10A. In the hanging drop culture, MDA-MB-231/HER+ generated compact well-rounded spheroids, while MCF-10A failed to form compact cellular masses. The multi-targeted nanosystem showed much better uptake, higher PE38 expression, and subsequent cell death in MDA-MB-231/HER2+ compared to MCF-10A. However, the efficacy of our targeted toxin gene therapy was lower in MDA-MB-231/HER2+ spheroids compared with that in the monolayer culture. the combination of the cell surface, transcriptional, and translational targeting increased the stringency of the treatment.

scholarly journals Combined transcriptional, translational and cell surface targeted gene therapy of HER2-positive breast cancer stem cells in three-dimentional cell culture

2021 ◽  
Author(s):  
Cobra Moradian ◽  
Fatemeh Rahbarizadeh
Keyword(s):  
Breast Cancer ◽  
Stem Cells ◽  
Gene Therapy ◽  
Growth Factor ◽  
Cancer Stem Cells ◽  
Monolayer Culture ◽  
Breast Cancer Stem Cells ◽  
Hanging Drop ◽  
Her2 Positive ◽  
Targeted Gene Therapy

Abstract Purpose Breast Cancer Stem Cells (BCSCs) resist conventional treatments and cause tumor recurrence. Almost 25% of breast cancers overexpress human epidermal growth factor receptor-2 (HER2). Here we developed a novel multi-targeted nanosystem to specifically eradicate HER2-positive BCSCs. Methods Plasmids containing CXCR1 promoter, PE38 toxin and 5′UTR of the basic fibroblast growth factor-2 (bFGF 5'UTR) were constructed. Polyamidoamine (PAMAM) dendrimers functionalized with an anti-HER2 VHH were used for plasmid delivery. Stem proportion of MCF-10A and MDA-MB-231/HER2+ (a cellular model of HER2 overexpression, developed in our lab) were evaluated by mammosphere formation assay. Hanging drop technique was used to produce spheroids. The uptake, gene expression and killing efficacy of the multi-targeted nanosystem were evaluated in both monolayer and spheroid culture. Results MDA-MB-231/HER2+ had more than 3 times higher ability to form mammosphere compared to MCF-10A, representing higher stem proportion in MDA-MB-231/HER2+. In hanging drop culture, MDA-MB-231/HER+ generated compact well-rounded spheroids, while MCF-10A failed to form compact cellular masses. The multi-targeted nanosystem showed much better uptake, higher PE38 expression and subsequent cell death in MDA-MB-231/HER2+ compared to MCF-10A. However efficacy of our targeted toxin gene therapy were lower in MDA-MB-231/HER2+ spheroid than those in monolayer. Conclusions Our novel multi-targeted nanosystem resulted in synergistic cytotoxic effects in HER2-positive BCSCs. This results demonstrate the necessity to use a combinatorial tumor targeting approach, to restrict the expression of a killer gene to target cells. Lower efficacy of our targeted gene therapy in spheroid models vs. monolayer culture suggest that, anticancer therapy assessments using spheroid might be more predictive of clinical efficacy than conventional monolayer culture.

scholarly journals Dysregulation of Transcription Factor EB Contributes to Cell Proliferation, Metastasis and Stemness in Breast Cancer

2021 ◽  
Author(s):  
Ningwei Fu ◽  
Ning Fan ◽  
Wenchao Luo ◽  
Lijia Lv ◽  
Jing Li ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Stem Cells ◽  
Cancer Stem Cells ◽  
Cancer Cells ◽  
Breast Cancer Cells ◽  
Migration And Invasion ◽  
Breast Cancer Stem Cells ◽  
Cancer Proliferation ◽  
Mammosphere Formation

Abstract Purpose: TFEB is a key regulator of autophagy-lysosomal biogenesis pathways, while its dysregulation is highly prevalent in various human cancers, but the specific contribution to breast cancer remains poorly understood. The main purpose of this study is to explore the role of TFEB in breast cancer proliferation, metastasis and maintaining breast cancer stem cells (BCSCs) traits, thus uncovering its underlying mechanism.Methods: Bioinformatics, western blotting and immunohistochemical staining were applied to analyze the expression of TFEB in breast cancer. Stable down-regulation TFEB cells were established in MCF-7 and MDA-MB-231 breast cancer cell lines. MTT, clone formation, wound healing, transwell and 3D tumor invasion assays were used to evaluate the proliferation, migration and invasion ability of breast cancer cells. Mammosphere formation, immunocytochemical (ICC) staining were used to detect the effect of down-regulating TFEB on breast cancer stem cells. Results: we demonstrated that higher expression of TFEB was found in breast cancer. TFEB depletion had inhibitory effects on cellular proliferation, migration and invasion of breast cancer cells. Moreover, knockdown TFEB decreased mammosphere formation ability of BCSCs and expression of cancer stem cell markers. Autophagy-lysosomal related proteins were decreased by down regulation of TFEB. Conclusion: we uncovered a critical role of TFEB in breast cancer proliferation and metastasis, and BCSCs self-renewal and stemness. The underlying mechanisms involve in maintaining BCSCs traits, and dysregulating lysosome functions.

scholarly journals Breast fibroblasts in both cancer and normal tissues induce phenotypic transformation of breast cancer stem cells: a preliminary study

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e4805 ◽  
Author(s):  
Bixiao Wang ◽  
Chunfang Xi ◽  
Mingwei Liu ◽  
Haichen Sun ◽  
Shuang Liu ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Stem Cells ◽  
Cancer Stem Cells ◽  
Conditioned Medium ◽  
Cell Invasion ◽  
Epithelial Mesenchymal Transition ◽  
Breast Cancer Stem Cells ◽  
Mesenchymal Transition ◽  
Mammosphere Formation ◽  
Phenotypic Transformation

Background Breast cancer stem cells (BCSCs) are associated with the invasion of breast cancer. In recent years, studies have demonstrated different phenotypes among BCSCs. Furthermore, BCSCs of diverse phenotypes are present at different tumour sites and different histological stages. Fibroblasts are involved in the phenotypic transformation of BCSCs. Cancer-associated fibroblasts (CAFs) participate in the induction of epithelial–mesenchymal transition, thereby promoting the acquisition of stem cell characteristics, but little is known about the role of normal fibroblasts (NFs) in the phenotypic transformation of BCSCs or about the effect of CAFs and NFs on BCSC phenotypes. Methods A total of six pairs of primary CAFs and NFs were isolated from surgical samples of breast cancer patients and subjected to morphological, immunohistochemical, cell invasion and proteomics analyses. After establishing a cell culture system with conditioned medium from CAFs and NFs, we used the mammosphere formation assay to explore the effect of CAFs and NFs on the self-renewal ability of BCSCs. The effect of CAFs and NFs on the phenotypic differentiation of BCSCs was further analysed by flow cytometry and immunofluorescence. Results The isolated CAFs and NFs did not show significant differences in cell morphology or alpha-smooth muscle actin (α-SMA) expression, but cell invasion and proteomics analyses demonstrated heterogeneity among these fibroblasts. Both CAFs and NFs could promote the generation of BCSCs, but CAFs displayed a greater ability than NFs in promoting mammosphere formation. Conditioned medium from CAFs increased the proportion of aldehyde dehydrogenase-1 positive (ALDH1+) BCSCs, but conditioned medium from NFs was more likely to promote the generation of CD44+CD24− BCSCs from MCF-7 cells. Discussion This study validated the heterogeneity among CAFs and NFs and expanded on the conclusion that fibroblasts promote the generation of cancer stem cells. Our results particularly emphasized the effect of NFs on the phenotypic transformation of BCSCs. In addition, this study further highlighted the roles of CAFs and NFs in the induction of different phenotypes in BCSCs.

scholarly journals O-Acetyl-GD2 as a Therapeutic Target for Breast Cancer Stem Cells

2022 ◽  
Vol 12 ◽  
Author(s):  
Jing-Yan Cheng ◽  
Jung-Tung Hung ◽  
Juway Lin ◽  
Fei-Yun Lo ◽  
Jing-Rong Huang ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Stem Cells ◽  
Cancer Stem Cells ◽  
Tumor Growth ◽  
Dependent Manner ◽  
Breast Cancer Stem Cells ◽  
Lipid Molecule ◽  
Mammosphere Formation

SynopsisA sugar-lipid molecule called OAcGD2 is a novel marker for breast cancer stem cells. Treatment with anti-OAcGD2 mAb8B6 may have superior anticancer efficacy by targeting cancer stem cells, thereby reducing metastasis and recurrence of cancer.BackgroundCancer stem cells (CSCs) that drive tumor progression and disease recurrence are rare subsets of tumor cells. CSCs are relatively resistant to conventional chemotherapy and radiotherapy. Eradication of CSCs is thus essential to achieve durable responses. GD2 was reported to be a CSC marker in human triple-negative breast cancer, and anti-GD2 immunotherapy showed reduced tumor growth in cell lines. Using a specific anti-OAcGD2 antibody, mAb8D6, we set out to determine whether OAcGD2+ cells exhibit stem cell properties and mAb8D6 can inhibit tumor growth by targeting OAcGD2+CSCs.MethodOAcGD2 expression in patient-derived xenografts (PDXs) of breast cancer was determined by flow cytometric analyses using mAb8D6. The stemness of OAcGD2+ cells isolated by sorting and the effects of mAb8B6 were assessed by CSC growth and mammosphere formation in vitro and tumor growth in vivo using PDX models.ResultWe found that the OAcGD2 expression levels in six PDXs of various molecular subtypes of breast cancer highly correlated with their previously defined CSC markers in these PDXs. The sorted OAcGD2+ cells displayed a greater capacity for mammosphere formation in vitro and tumor initiation in vivo than OAcGD2− cells. In addition, the majority of OAcGD2+ cells were aldehyde dehydrogenase (ALDH+) or CD44hiCD24lo, the known CSC markers in breast cancer. Treatment of PDXs-bearing mice with mAb8B6, but not doxorubicin, suppressed the tumor growth, along with reduced CSCs as assessed by CSC markers and in vivo tumorigenicity. In vitro, mAb8B6 suppressed proliferation and mammosphere formation and induced apoptosis of OAcGD2+ breast cancer cells harvested from PDXs, in a dose-dependent manner. Finally, administration of mAb8B6 in vivo dramatically suppressed tumor growth of OAcGD2+ breast CSCs (BCSCs) with complete tumor abrogation in 3/6 mice.ConclusionOAcGD2 is a novel marker for CSC in various subtypes of breast cancer. Anti-OAcGD2 mAb8B6 directly eradicated OAcGD2+ cells and reduced tumor growth in PDX model. Our data demonstrate the potential of mAb8B6 as a promising immunotherapeutic agent to target BCSCs.

scholarly journals Machilin D, a Lignin Derived from Saururus chinensis, Suppresses Breast Cancer Stem Cells and Inhibits NF-κB Signaling

Biomolecules ◽  
2020 ◽  
Vol 10 (2) ◽  
pp. 245 ◽  
Author(s):  
Xing Zhen ◽  
Hack Sun Choi ◽  
Ji-Hyang Kim ◽  
Su-Lim Kim ◽  
Ren Liu ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Stem Cells ◽  
Cancer Stem Cells ◽  
High Performance ◽  
Reversed Phase ◽  
Breast Cancer Stem Cells ◽  
Mammosphere Formation ◽  
Saururus Chinensis ◽  
Isolated Compound ◽  
Layer Chromatography

Cancer stem cells are responsible for breast cancer initiation, metastasis, and relapse. Targeting breast cancer stem cells (BCSCs) using phytochemicals is a good strategy for the treatment of cancer. A silica gel, a reversed-phase C18 column (ODS), a Sephadex LH-20 gel, thin layer chromatography, and high-performance liquid chromatography (HPLC) were used for compound isolation from Saururus chinensis extracts. The isolated compound was identified as machilin D by mass spectrometry and nuclear magnetic resonance (NMR). Machilin D inhibited the growth and mammosphere formation of breast cancer cells and inhibited tumor growth in a xenograft mouse model. Machilin D reduced the proportions of CD44+/CD24- and aldehyde dehydrogenase 1 (ALDH1)-positive cells. Furthermore, this compound reduced the nuclear localization of the NF-κB protein and decreased the IL-6 and IL-8 secretion in mammospheres. These results suggest that machilin D blocks IL-6 and IL-8 signaling and induces CSC death and thus may be a potential agent targeting BCSCs.

scholarly journals Propofol Reduced Mammosphere Formation of Breast Cancer Stem Cells via PD-L1/Nanog In Vitro

2019 ◽  
Vol 2019 ◽  
pp. 1-9 ◽  
Author(s):  
Xiaobei Zhang ◽  
Fangxuan Li ◽  
Ying Zheng ◽  
Xiaokun Wang ◽  
Kaiyuan Wang ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Stem Cells ◽  
Cell Proliferation ◽  
Cancer Stem Cells ◽  
Cancer Recurrence ◽  
Tumor Formation ◽  
Breast Cancer Stem Cells ◽  
Mammosphere Formation ◽  
Significant Difference

Several researches revealed that propofol, a hypnotic intravenous anesthesia agent, could inhibit the cancer cell proliferation and tumor formation, which might affect cancer recurrence or metastasis and impact patients’ prognosis. Cancer stem cells (CSCs) comprised a tiny fraction of tumor bulk and played a vital role in cancer recurrence and eventual mortality. This study investigates the effect of propofol on breast cancer stem cells (BCSCs) in vitro and the underlying molecular mechanisms. Tumor formation of CSCs was measured by mammosphere culture. Cultured BCSCs were exposed to different concentrations and durations of propofol. Cell proliferation and self-renewal capacity were determined by MTT assays. Expressions of PD-L1 and Nanog were measured using western blotting and real-time PCR. We knocked down the PD-L1 expression in MDA-MB-231 cells by lentivirus-mediated RNAi technique, and the mammosphere-forming ability of shControl and shPD-L1 under propofol treatment was examined. Mammosphere culture could enrich BCSCs. Compared with control, cells exposed to propofol for 24 h induced a larger number of mammosphere cells (P=0.0072). Levels of PD-L1 and Nanog were downregulated by propofol. Compared with shControl stem cells, there was no significant difference in the inhibitory effect of propofol on the mammosphere-forming ability of shPD-L1 stem cells which indicated that the inhibition of propofol could disappear in PD-L1 knockdown breast stem cells. Propofol could reduce the mammosphere-forming ability of BCSCs in vitro. Mechanism experiments indicated that the inhibition of propofol in mammosphere formation of BCSCs might be mediated through PD-L1, which was important to maintain Nanog.

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