CD44 Targeting Mediated by Polymeric Nanoparticles and Combination of Chlorine TPCS2a-PDT and Docetaxel-Chemotherapy for Efficient Killing of Breast Differentiated and Stem Cancer Cells In Vitro.
ABSTRACT: The presence of rare but highly tumorigenic cancer stem cells (CSCs) within the tumors is recognized as one of the major reasons of failure of conventional chemotherapies, mainly attributed to the development of drug resistance and increasing metastatic potential. Here, we propose a therapeutic strategy based on the simultaneous delivery of docetaxel (DTX) and the photosensitizer meso-tetraphenyl chlorine disulfonate (TPCS2a) using hyaluronic acid (HA) coated polymeric nanoparticles (HA-NPs) for the targeting and killing of CD44 over-expressing breast cancer (BC) cells, both differentiated and CSCs (CD44high/CD24low population), thus combining chemotherapy and photodynamic therapy (PDT). Using the CD44high MDA-MB-231 and the CD44low MCF-7 cells, we demonstrated the occurrence of CD44-mediated uptake of HA-NPs both in monolayers and mammosphere cultures enriched in CSCs. Cell treatments showed that combination therapy using co-loaded NPs (HA@DTX/TPCS2a-NPs) had superior efficacy over monotherapies (HA@DTX-NPs or HA@TPCS2a-NPs) in reducing the self-renewal capacity, measured as mammosphere formation efficiency, and in eradicating the CSC population evaluated with aldehyde dehydrogenase activity assay and CD44/CD24 immunostaining. In summary, these in vitro studies demonstrated for the first time the potential of the combination of DTX-chemotherapy and TPCS2a-PDT for killing CSCs using properly designed NPs.
Project description:<h4>Background</h4>Novel polymeric nanoparticles (NPs) specifically designed for delivering chemotherapeutics in the body and aimed at improving treatment activity and selectivity, cover a very relevant area in the field of nanomedicine. Here, we describe how to build a polymer shell of Hyaluronan (HA) and Polyethyleneimine (PEI) on biodegradable NPs of poly(lactic-co-glycolic) acid (PLGA) through electrostatic interactions and to achieve NPs with unique features of sustained delivery of a docetaxel (DTX) drug cargo as well as improved intracellular uptake.<h4>Results</h4>A stable PEI or HA/PEI shell could be obtained by careful selection of layering conditions. NPs with exquisite stability in salt and protein-rich media, with size and surface charge matching biological requirements for intravenous injection and endowed with sustained DTX release could be obtained. Cytotoxicity, uptake and activity of both PLGA/PEI/HA and PLGA/PEI NPs were evaluated in CD44(+) (A549) and CD44(-) (Calu-3) lung cancer cells. In fact, PEI-coated NPs can be formed after degradation/dissociation of the surface HA because of the excess hyaluronidases overexpressed in tumour interstitium. There was no statistically significant cytotoxic effect of PLGA/PEI/HA and PLGA/PEI NPs in both cell lines, thus suggesting that introduction of PEI in NP shell was not hampered by its intrinsic toxicity. Intracellular trafficking of NPs fluorescently labeled with Rhodamine (RHO) (RHO-PLGA/PEI/HA and RHO-PLGA/PEI NPs) demonstrated an increased time-dependent uptake only for RHO-PLGA/PEI/HA NPs in A549 cells as compared to Calu-3 cells. As expected, RHO-PLGA/PEI NP uptake in A549 cells was comparable to that observed in Calu-3 cells. RHO-PLGA/PEI/HA NPs internalized into A549 cells showed a preferential perinuclear localization. Cytotoxicity data in A549 cells suggested that DTX delivered through PLGA/PEI/HA NPs exerted a more potent antiproliferative activity than free DTX. Furthermore, DTX-PLGA/PEI NPs, as hypothetical result of hyaluronidase-mediated degradation in tumor interstitium, were still able to improve the cytotoxic activity of free DTX.<h4>Conclusions</h4>Taken together, results lead us to hypothesize that biodegradable NPs coated with a PEI/HA shell represent a very promising system to treat CD44 overexpressing lung cancer. In principle, this novel nanocarrier can be extended to different single drugs and drug combinations taking advantage of the shell and core properties.
Project description:Cancer stem cells (CSCs) are innately resistant to standard therapies, which positions CSCs in the focus of anti-cancer research. In this study, we investigated the potential inhibitory effect of tannic acid (TA) on CSCs. Our data demonstrated that TA (10 μM), at the concentration not inhibiting the proliferation of normal mammary cells (MCF10A), inhibited the formation and growth of mammosphere in MCF7, T47D, MDA-MB-231 cells shown as a decrease in mammosphere formation efficiency (MFE), cell number, diameter of mammosphere, and ALDH1 activity. NF-κB pathway was activated in the mammosphere indicated by an up-regulation of p65, a degradation of IκBα, and an increased IL-6. The inhibition of NF-κB pathway via gene silencing of p65 (sip65), NF-κB inhibitor (PDTC), or IKK inhibitor (Bay11-7082) alleviated MFE. Other CSCs markers such as an increase in ALDH1 and CD44high/CD24low ratio were ameliorated by sip65. TA also alleviated TGFβ-induced EMT, increase in MFE, and NF-κB activation. In murine xenograft model, TA reduced tumor volume which was associated with a decrease in CD44high/CD24low expression and IKK phosphorylation. These results suggest that TA negatively regulates CSCs by inhibiting NF-κB activation and thereby prevents cancer cells from undergoing EMT and CSCs formation, and may thus be a promising therapy targeting CSCs.
Project description:Cancer stem cells have certain characteristics, such as self-renewal, differentiation, and drug resistance, which are related to tumor progression, maintenance, recurrence, and metastasis. In our study, we targeted breast cancer stem cells (BCSCs) using a natural compound, coriolic acid, from Salicornia herbacea L. This compound was isolated by mammosphere formation inhibition bioassay-guided fractionation and identified by using NMR spectroscopy and electrospray ionization mass spectrometry. Coriolic acid inhibited the formation of mammospheres and induced BCSC apoptosis. It also decreased the subpopulation of CD44high/CD24low cells, a cancer stem cell (CSC) phenotype, and specific genes related to CSCs, such as Nanog,Oct4, and CD44. Coriolic acid decreased the transcriptional and translational levels of the c-Myc gene, which is a CSC survival factor. These results indicated that coriolic acid could be a novel compound to target BCSCs via regulation of c-Myc.
Project description:<h4>Background</h4>Cancer stem cells (CSCs) are highly proliferative and tumorigenic, which contributes to chemotherapy resistance and tumor occurrence. CSCs specific therapy may achieve excellent therapeutic effects, especially to the drug-resistant tumors.<h4>Results</h4>In this study, we developed a kind of targeting nanoparticle system based on cationic albumin functionalized with hyaluronic acid (HA) to target the CD44 overexpressed CSCs. All-trans-retinoic acid (ATRA) was encapsulated in the nanoparticles with ultrahigh encapsulation efficiency (EE%) of 93% and loading content of 8.37%. TEM analysis showed the nanoparticles were spherical, uniform-sized and surrounded by a coating layer consists of HA. Four weeks of continuously measurements of size, PDI and EE% revealed the high stability of nanoparticles. Thanks to HA conjugation on the surface, the resultant nanoparticles (HA-eNPs) demonstrated high affinity and specific binding to CD44-enriched B16F10 cells. In vivo imaging revealed that HA-eNPs can targeted accumulate in tumor-bearing lung of mouse. The cytotoxicity tests illustrated that ATRA-laden HA-eNPs possessed better killing ability to B16F10 cells than free drug or normal nanoparticles in the same dose, indicating its good targeting property. Moreover, HA-eNPs/ATRA treatment decreased side population of B16F10 cells significantly in vitro. Finally, tumor growth was significantly inhibited by HA-eNPs/ATRA in lung metastasis tumor mice.<h4>Conclusions</h4>These results demonstrate that the HA functionalized albumin nanoparticles is an efficient system for targeted delivery of antitumor drugs to eliminate the CSCs.
Project description:<h4>Introduction</h4>Triple-negative breast cancer (TNBC) high rate of relapse is thought to be due to the presence of tumor-initiating cells (TICs), molecularly defined as being CD44high/CD24-/low. TICs are resilient to chemotherapy and radiation. However, no currently accepted molecular target exists against TNBC and, moreover, TICs. Therefore, we sought the identification of kinase targets that inhibit TNBC growth and eliminate TICs.<h4>Methods</h4>A genome-wide human kinase small interfering RNA (siRNA) library (691 kinases) was screened against the TNBC cell line SUM149 for growth inhibition. Selected siRNAs were then tested on four different breast cancer cell lines to confirm the spectrum of activity. Their effect on the CD44high subpopulation and sorted CD44high/CD24-/low cells of SUM149 also was studied. Further studies were focused on polo-like kinase 1 (PLK1), including its expression in breast cancer cell lines, effect on the CD44high/CD24-/low TIC subpopulation, growth inhibition, mammosphere formation, and apoptosis, as well as the activity of the PLK1 inhibitor, BI 2536.<h4>Results</h4>Of the 85 kinases identified in the screen, 28 of them were further silenced by siRNAs on MDA-MB-231 (TNBC), BT474-M1 (ER+/HER2+, a metastatic variant), and HR5 (ER+/HER2+, a trastuzumab-resistant model) cells and showed a broad spectrum of growth inhibition. Importantly, 12 of 28 kinases also reduced the CD44high subpopulation compared with control in SUM149. Further tests of these 12 kinases directly on a sorted CD44high/CD24-/low TIC subpopulation of SUM149 cells confirmed their effect. Blocking PLK1 had the greatest growth inhibition on breast cancer cells and TICs by about 80% to 90% after 72 hours. PLK1 was universally expressed in breast cancer cell lines, representing all of the breast cancer subtypes, and was positively correlated to CD44. The PLK1 inhibitor BI 2536 showed similar effects on growth, mammosphere formation, and apoptosis as did PLK1 siRNAs. Finally, whereas paclitaxel, doxorubicin, and 5-fluorouracil enriched the CD44high/CD24-/low population compared with control in SUM149, subsequent treatment with BI 2536 killed the emergent population, suggesting that it could potentially be used to prevent relapse.<h4>Conclusion</h4>Inhibiting PLK1 with siRNA or BI 2536 blocked growth of TNBCs including the CD44high/CD24-/low TIC subpopulation and mammosphere formation. Thus, PLK1 could be a potential therapeutic target for the treatment of TNBC as well as other subtypes of breast cancer.
Project description:BACKGROUND:The enrichment of cancer stem cell-like cells (CSCs) has been considered to be responsible for tumor progression after an initial response to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (EGFR-TKIs) in patients with non-small cell lung adenocarcinoma (NSCLC/ADC). CSCs with ALDH1A1bright /CD44high expression contribute to the TKIs resistance in NSCLC/ADC cells. All-trans retinoic acid (ATRA) has been shown to be a potential targeted therapy against CSCs due to its ability to inhibit ALDH1A1 activity. We therefore investigated whether ATRA could circumvent the resistance to improve the response to gefitinib in NSCLC/ADC cells. METHODS:Treatment of NSCLC/ADC A549 and H1650 cells with gefitinib enriched the gefitinib surviving cells (GSCs). The expression of ALDH1A1 and CD44 and the IC50 values for gefitinib were determined by flow cytometry (FCM) and crystal violet assay in GSCs and ATRA-treated GSCs, respectively. Using DEAB as the positive control, direct inhibitory effect of ATRA on ALDH1A1 activity was determined by ALDEFLUOR assay, RESULTS: GSCs showed higher expression of ALDH1A1 and CD44 and IC50 values for gefitinib than their respective parental cells, suggesting that gefitinib can lead to propagation of CSC-enriched gefitinib-resistant cells. Treatment with ATRA was found to significantly reduce the increased expression of ALDH1A1 and CD44 and the IC50 values for gefitinib in A549GSC and H1650GSC cells, and ATRA could directly inhibit active ALDH1A1 as compared to DEAB. CONCLUSION:Our findings suggest that combination treatment with ATRA prevents gefitinib-induced enrichment of ALDH1A1bright/CD44high CSCs and enhances gefitinib-induced growth inhibition of NSCLC/ADC cells.
Project description:The purpose of this study was to determine association between cancer stem cells (CSCs) and their niche with progression of oral potentially malignant disorders.Patients with histologically confirmed oral potentially malignant disorders, stratified into high/low risk lesions based on the degree of dysplasia and oral cancer were included in this study. Immunohistochemical profiling of markers of CSCs (CD44), endothelial cells (CD31) and CSC-vascular niche cross-talk (CXCR4 and SDF1) were carried out. Statistical analysis was performed to correlate the relationship of markers with histopathology grade (ANOVA, and ?2 test, unpaired t test) using GraphPad InStat v3.06.The study included 550 samples (349 patients) and analysis showed progressive increase in expression levels of CSC and its niche markers with increase in grade of dysplasia as compared to the normal cohort (p?<?0.05). Co-expression analysis revealed that, in comparison to the normal cohort, a larger percentage of patients showed increased expression of CD31 and CD44 (CD31high/CD44high; p?<?0.05) and of CXCR4 and SDF1 (CXCR4high/SDF1high; p?=?0.04), suggesting an association of the CSCs and the vascular niche. Further, distribution of patients with CD44high/CXCR4high (p?<?0.05) and CD31high/SDF1high (p?=?0.01) was significantly increased in the high-risk group (18%), suggesting a correlation between CD44+/CXCR4+ cells, the vascular niche and progression of oral dysplastic lesions.The increased expression of CSCs, the vascular niche and their cross talk markers are associated with increase in severity of dysplasia suggesting their role in the progression of oral potentially malignant disorders and may hence be used in identifying high-risk OPMD.
Project description:Photo-immunotherapy is a novel therapeutic approach against malignant tumors with minimal invasiveness. Herein, a targeting multifunctional black phosphorus (BP) nanoparticle, modified by PEGylated hyaluronic acid (HA), was designed for photothermal/photodynamic/photo-immunotherapy. In vitro and in vivo assays indicated that HA-BP nanoparticles possess excellent biocompatibility, stability, and sufficient therapeutic efficacy in the combined therapy of photothermal therapy (PTT) and photodynamic therapy (PDT) for cancer therapy. Moreover, the results of in vitro showed that HA-BP down-regulated the expression of CD206 (M2 macrophage marker) by 42.3% and up-regulated the ratio of CD86?M1 macrophage marker?by 59.6%, indicating that HA-BP nanoparticles have functions in remodeling tumor associated macrophages (TAMs) phenotype (from pro-tumor M2 TAMs to anti-tumor M1 macrophages). Fluorescence (FL) and photoacoustic (PA) multimodal imaging confirmed the selective accumulation of HA-BP in tumor site via both CD44+ mediated active targeting and passive EPR effect. In vitro and in vivo studies suggested that the combined therapy of PDT, PTT and immunotherapy using HA-BP could not only significantly inhibit original tumor but also induce immunogenic cell death (ICD) and release Damage-associated molecular patterns (DAMPs), which could induce maturation of dendritic cells (DCs) and activate effector cells that robustly evoke the antitumor immune responses for cancer treatment. This study expands the biomedical application of BP nanoparticles and displays the potential of modified BP as a multifunctional therapeutic platform for the future cancer therapy. Graphical abstract Image 1 Highlights • Novel HA-BP NPs were synthesized for photothermal/photodynamic/photo-immunotherapy.• HA-BP NPs with size around 50 nm possess remarkable enhancement of tumor targeting and accumulation ability.• HA-BP NPs could promote M2 to M1 macrophage polarization and further enhance invasion of activated CD4+ and CD8+ T cells.
Project description:Purpose:Chemotherapy of colon cancer needs improvement to mitigate the severe adverse effects (AEs) associated with the cytotoxic drugs. The aim of this study is to develop a novel targeted drug delivery system (TDDS) with practical application potential for colon cancer treatment. Methods:The TDDS was built by loading docetaxel (DTX) in albumin nanoparticles (NPs) that were functionalized with nucleolin-targeted aptamers (AS1411). Results:The TDDS (Apt-NPs-DTX) had an average size of 62 nm and was negatively charged with a zeta potential of -31.2 mV. DTX was released from the albumin NP with a typical sustained release profile. Aptamer-guided NPs were preferentially ingested by nucleolin-expressing CT26 colon cancer cells vs the control cells. In vitro cytotoxicity study showed that Apt-NPs-DTX significantly enhanced the killing of CT26 colon cancer cells. Importantly, compared with non-targeted drug delivery, Apt-NPs-DTX treatment significantly improved antitumor efficacy and prolonged the survival of CT26-bearing mice, without raising systemic toxicity. Conclusion:The results suggest that Apt-NPs-DTX has potential in the targeted treatment of colon cancer.