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Effects of CS-binding peptide (C6S-p) on glioma cells. (A) Immunocytochemistry of biotin staining with streptavidin-Alexa 594 (red) and nuclear with DAPI (blue). A172 cells were treated with PBS or C6S-p for 20 min or C6S-p for
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Chondroitin sulfate (CS) is a major component of the extracellular matrix found to be abnormally accumulated in several types of cancer tissues. Previous studies have indicated that CS synthases and modification enzymes are frequently elevated in human gliomas and are associated with poor prognosis. However, the underlying mechanisms of CS in cance...
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Context 1
... tested a C6S-specific binding peptide (C6S-p, biotinylated), which revealed high affinity to C6S and rescued the inhibition of C6S on neurite outgrowth. Our in vitro studies showed that C6S-p binds to the glioma cell surface under culture conditions ( Figure 5A). Subsequent assessment of cell viability to evaluate the cytotoxicity of C6S-p using the CCK8 assay revealed that C6S-p treatment slightly decreased, but did not significant affect, the viability of A172 cells in both dose-and time-dependent experiments ( Figure 5B). ...
Context 2
... in vitro studies showed that C6S-p binds to the glioma cell surface under culture conditions ( Figure 5A). Subsequent assessment of cell viability to evaluate the cytotoxicity of C6S-p using the CCK8 assay revealed that C6S-p treatment slightly decreased, but did not significant affect, the viability of A172 cells in both dose-and time-dependent experiments ( Figure 5B). To determine the effect of C6S-p on the mobility of glioma cells, scratch healing assays were performed. ...
Context 3
... determine the effect of C6S-p on the mobility of glioma cells, scratch healing assays were performed. The results of these assays showed that the C6S binding peptide inhibited the cell migration for recovering the scratch in both GL261 and A172 cells as compared to the scramble peptide control; the recovered area in the scramble peptide control treatment vs. C6S binding peptide treatment was 50% versus 25% for the GL261 cells and 91% versus 51% for the A172 cells, respectively ( Figure 5C). Consistent inhibitory effects of C6S-p were also observed in the transwell invasion assays ( Figure 5D). ...
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... results of these assays showed that the C6S binding peptide inhibited the cell migration for recovering the scratch in both GL261 and A172 cells as compared to the scramble peptide control; the recovered area in the scramble peptide control treatment vs. C6S binding peptide treatment was 50% versus 25% for the GL261 cells and 91% versus 51% for the A172 cells, respectively ( Figure 5C). Consistent inhibitory effects of C6S-p were also observed in the transwell invasion assays ( Figure 5D). Further assessment of the effects of CS on ex vivo CNS tissues was performed using the brain slice migration assays. ...
Context 5
... assessment of the effects of CS on ex vivo CNS tissues was performed using the brain slice migration assays. The results revealed that both the CHSY1 knockdown and C6S-p treatment groups significantly suppressed the migration area of CNS cells in the brain slice migration assays ( Figure 5E). ...
Context 6
... tested a C6S-specific binding peptide (C6S-p, biotinylated), which revealed high affinity to C6S and rescued the inhibition of C6S on neurite outgrowth. Our in vitro studies showed that C6S-p binds to the glioma cell surface under culture conditions ( Figure 5A). Subsequent assessment of cell viability to evaluate the cytotoxicity of C6S-p using the CCK8 assay revealed that C6S-p treatment slightly decreased, but did not significant affect, the viability of A172 cells in both doseand time-dependent experiments ( Figure 5B). ...
Context 7
... in vitro studies showed that C6S-p binds to the glioma cell surface under culture conditions ( Figure 5A). Subsequent assessment of cell viability to evaluate the cytotoxicity of C6S-p using the CCK8 assay revealed that C6S-p treatment slightly decreased, but did not significant affect, the viability of A172 cells in both doseand time-dependent experiments ( Figure 5B). To determine the effect of C6S-p on the mobility of glioma cells, scratch healing assays were performed. ...
Context 8
... determine the effect of C6S-p on the mobility of glioma cells, scratch healing assays were performed. The results of these assays showed that the C6S binding peptide inhibited the cell migration for recovering the scratch in both GL261 and A172 cells as compared to the scramble peptide control; the recovered area in the scramble peptide control treatment vs. C6S binding peptide treatment was 50% versus 25% for the GL261 cells and 91% versus 51% for the A172 cells, respectively ( Figure 5C). Consistent inhibitory effects of C6S-p were also observed in the transwell invasion assays ( Figure 5D). ...
Context 9
... results of these assays showed that the C6S binding peptide inhibited the cell migration for recovering the scratch in both GL261 and A172 cells as compared to the scramble peptide control; the recovered area in the scramble peptide control treatment vs. C6S binding peptide treatment was 50% versus 25% for the GL261 cells and 91% versus 51% for the A172 cells, respectively ( Figure 5C). Consistent inhibitory effects of C6S-p were also observed in the transwell invasion assays ( Figure 5D). Further assessment of the effects of CS on ex vivo CNS tissues was performed using the brain slice migration assays. ...
Context 10
... assessment of the effects of CS on ex vivo CNS tissues was performed using the brain slice migration assays. The results revealed that both the CHSY1 knockdown and C6S-p treatment groups significantly suppressed the migration area of CNS cells in the brain slice migration assays ( Figure 5E). ...
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Citations
... Prior studies have shown that CHPF increases C6S on cancer cells [12,44], and the C6S-specific binding peptide (C6S-p) identified from a phage display peptide library has been proposed to block certain C6S biological functions, such as rescuing neurite outgrowth and reducing glioma cell invasion [24,25,45,46]. Thus, we investigated whether C6S-p could target CS-SDC1 on breast cancer cells. ...
... However, only a few studies have examined the methods for blocking C6S bioactivities in cancer treatment. In a previous study, a C6S-specific binding peptide was used on cultured glioblastoma cells, and the treatment suppressed glioma cell mobility accompanied by promoting CD44 degradation, but its effects were not evaluated in vivo [45]. Dr Karumbaiah reported that Surfen could block extratumoral CS and inhibit the invasion of F98 (a rat GBM cell line) cells, but the therapeutic effects of Surfen are not significant in animal models [58]. ...
Accumulation of abnormal chondroitin sulfate (CS) chains in breast cancer tissue is correlated with poor prognosis. However, the biological functions of these CS chains in cancer progression remain largely unknown, impeding the development of targeted treatment focused on CS. Previous studies identified chondroitin polymerizing factor (CHPF; also known as chondroitin sulfate synthase 2) is the critical enzyme regulating CS accumulation in breast cancer tissue. We then assessed the association between CHPF‐associated proteoglycans (PGs) and signaling pathways in breast cancer datasets. The regulation between CHPF and syndecan 1 (SDC1) was examined at both the protein and RNA levels. Confocal microscopy and image flow cytometry were employed to quantify macropinocytosis. The effects of the 6‐O‐sulfated CS‐binding peptide (C6S‐p) on blocking CS functions were tested in vitro and in vivo. Results indicated that the expression of CHPF and SDC1 was tightly associated within primary breast cancer tissue, and high expression of both genes exacerbated patient prognosis. Transforming growth factor beta (TGF‐β) signaling was implicated in the regulation of CHPF and SDC1 in breast cancer cells. CHPF supported CS–SDC1 stabilization on the cell surface, modulating macropinocytotic activity in breast cancer cells under nutrient‐deprived conditions. Furthermore, C6S‐p demonstrated the ability to bind CS‐SDC1, increase SDC1 degradation, suppress macropinocytosis of breast cancer cells, and inhibit tumor growth in vivo. Although other PGs may also be involved in CHPF‐regulated breast cancer malignancy, this study provides the first evidence that a CS synthase participates in the regulation of macropinocytosis in cancer cells by supporting SDC1 expression on cancer cells.
... Recent studies have reported that CHSY1 was overexpressed in various tumors (for example, hepatocellular carcinoma [24], glioma [25], soft tissue sarcomas [26]), demonstrating its possible association with tumor progression. Liu et al. [24] reported that CHSY1 could be involved in the induction of malignant behavior of cells through the regulation of hedgehog malignant behavior of hepatocellular carcinoma cells. ...
Background
The most common site of metastasis in colorectal cancer (CRC) is the liver and liver metastases occur in more than 50% of patients during diagnosis or treatment. The occurrence of metastasis depends on a series of events known as the invasive-metastasis cascade. Currently, the underlying genes and pathways regulating metastasis initiation in the liver microenvironment are unknown.
Methods
We performed systematic CRISPR/Cas9 screening using an in vivo mouse model of CRC liver metastasis to identify key regulators of CRC metastasis. We present the full results of this screen,which included a list of genes that promote or repress CRC liver colonization. By silencing these genes individually, we found that chondroitin sulfate synthase 1 (CHSY1) may be involved in CRC metastasis. We verified the function of CHSY1 and its involvement in liver metastasis of CRC through in vivo and in vitro experiments.
Result
The results of TCGA and CRISPR/Cas9 showed that CHSY1 was overexpressed in CRC primary and liver metastasis tissues and indicated a worse clinical prognosis. In vitro and in vivo experiments confirmed that CHSY1 facilitated the liver metastasis of CRC and CHSY1 induced CD8 ⁺ T cell exhaustion and upregulated PD-L1 expression. The metabolomic analysis indicated that CHSY1 promoted CD8 ⁺ T cell exhaustion by activating the succinate metabolism pathway leading to liver metastasis of CRC. Artemisinin as a CHSY1 inhibitor reduced liver metastasis and enhanced the effect of anti-PD1 in CRC. PLGA-loaded Artemisinin and ICG probe reduced liver metastasis and increased the efficiency of anti-PD1 treatment in CRC.
Conclusion
CHSY1 could promote CD8 ⁺ T cell exhaustion through activation of the succinate metabolic and PI3K/AKT/HIF1A pathway, leading to CRC liver metastasis. The combination of CHSY1 knockdown and anti-PD1 contributes to synergistic resistance to CRC liver metastasis. Artemisinin significantly inhibits CHSY1 activity and in combination with anti-PD1 could synergistically treat CRC liver metastases. This study provides new targets and specific strategies for the treatment of CRC liver metastases, bringing new hope and benefits to patients.
... Talin's role in facilitating traction force in cells is relatively well understood, given its direct role in focal adhesion dynamics, but CD44 is less studied. CD44 is an extracellular matrix receptor that connects to the actomyosin machinery via adapter proteins like integrins, and crosstalk with integrins has been reported 53,54 . The data suggest that CD44 may be more involved in the formation of nascent focal adhesions and initial cell adhesion/spreading and less in mature focal adhesions and cell contractility, supported by prior studies demonstrating that CD44KO does not disrupt focal adhesion formation 55 . ...
Mechanical forces drive critical cellular processes that are reflected in mechanical phenotypes, or mechanotypes, of cells and their microenvironment. We present here “Rupture And Deliver” Tension Gauge Tethers (RAD-TGTs) in which flow cytometry is used to record the mechanical history of thousands of cells exerting forces on their surroundings via their propensity to rupture immobilized DNA duplex tension probes. We demonstrate that RAD-TGTs recapitulate prior DNA tension probe studies while also yielding a gain of fluorescence in the force-generating cell that is detectable by flow cytometry. Furthermore, the rupture propensity is altered following disruption of the cytoskeleton using drugs or CRISPR-knockout of mechanosensing proteins. Importantly, RAD-TGTs can differentiate distinct mechanotypes among mixed populations of cells. We also establish oligo rupture and delivery can be measured via DNA sequencing. RAD-TGTs provide a facile and powerful assay to enable high-throughput mechanotype profiling, which could find various applications, for example, in combination with CRISPR screens and -omics analysis.
... This unique oncofetal chondroitin sulfate (ofCS) is omnipresent in malignancies, indicating an essential role in tumorigenesis. Indeed, several studies have reported increased levels of abnormally expressed chondroitin sulfate (CS) across various cancers, which correlates with poor prognosis [13][14][15][16]. CS is likewise found to inhibit TNF-α-induced NF-κB activation and downstream inflammatory processes [17,18]. ...
... Recovered mice stayed tumor-free and were partially protected against tumor rechallenge more than 70 days after the first cancer cell injection. Various glycosaminoglycans (hyaluronic acid, heparin) and proteoglycans (CD44, CSPG4, MUC-1) have been suggested as potential cancer targets due to their important roles in tumor pathogenesis and their upregulation in malignant tissues [14,16,18,50,51] but are also expressed in healthy tissues. This is also the case for chondroitin sulfate A in general. ...
Background
The malaria protein VAR2CSA binds oncofetal chondroitin sulfate (ofCS), a unique chondroitin sulfate, expressed on almost all mammalian cancer cells. Previously, we produced a bispecific construct targeting ofCS and human T cells based on VAR2CSA and anti-CD3 (V-aCD3 Hu ). V-aCD3 Hu showed efficacy against xenografted tumors in immunocompromised mice injected with human immune cells at the tumor site. However, the complex effects potentially exerted by the immune system as a result of the treatment cannot occur in mice without an immune system. Here we investigate the efficacy of V-aCD3 Mu as a monotherapy and combined with immune checkpoint inhibitors in mice with a fully functional immune system.
Methods
We produced a bispecific construct consisting of a recombinant version of VAR2CSA coupled to an anti-murine CD3 single-chain variable fragment. Flow cytometry and ELISA were used to check cell binding capabilities and the therapeutic effect was evaluated in vitro in a killing assay. The in vivo efficacy of V-aCD3 Mu was then investigated in mice with a functional immune system and established or primary syngeneic tumors in the immunologically “cold” 4T1 mammary carcinoma, B16-F10 malignant melanoma, the pancreatic KPC mouse model, and in the immunologically “hot” CT26 colon carcinoma model.
Results
V-aCD3 Mu had efficacy as a monotherapy, and the combined treatment of V-aCD3 Mu and an immune checkpoint inhibitor showed enhanced effects resulting in the complete elimination of solid tumors in the 4T1, B16-F10, and CT26 models. This anti-tumor effect was abscopal and accompanied by a systemic increase in memory and activated cytotoxic and helper T cells. The combined treatment also led to a higher percentage of memory T cells in the tumor without an increase in regulatory T cells. In addition, we observed partial protection against re-challenge in a melanoma model and full protection in a breast cancer model.
Conclusions
Our findings suggest that V-aCD3 Mu combined with an immune checkpoint inhibitor renders immunologically “cold” tumors “hot” and results in tumor elimination. Taken together, these data provide proof of concept for the further clinical development of V-aCD3 as a broad cancer therapy in combination with an immune checkpoint inhibitor.
... The decrease of versican levels when silencing Chsy1 is in agreement with previous studies which showed that the loss of CS-GAG from the tissue leads to CSPG degradation, such as versican, and CD44 [26,27]. Our results also revealed that β3-tubulin-positive axons gradually reappeared in the distal end of the recipient nerve after Chsy1-siRNA treatment, which was associated with the loss of versican V1 accumulation. ...
Versican is a chondroitin sulfate proteoglycan (CSPG), which deposits in perineurium as a physical barrier and prevents the growth of axons out of the fascial boundary. Several studies have indicated that the chondroitin sulfate (CS) chains on versican have several possible functions beyond the physical barrier, including the ability to stabilize versican core protein in the extracellular matrix. As chondroitin sulfate synthase 1 (Chsy1) is a crucial enzyme for CS elongation, we hypothesized that in vivo knockdown of Chsy1 at peripheral nerve lesion site may decrease CS and versican accumulation, and result in accelerating neurite regeneration. In the present study, end-to-side neurorrhaphy (ESN) in Wistar rats was used as an in vivo model of peripheral nerve injury to evaluate nerve regeneration after surgical intervention. The distribution and expression of versican and Chsy1 in regenerating axons after ESN was studied using confocal microscopy and western blotting. Chsy1 was silenced at the nerve lesion (surgical) site using in vivo siRNA transfection. The results indicated that Chsy1 was successfully silenced in nerve tissue, and its downregulation was associated with functional recovery of compound muscle action potential. Silencing of Chsy1 also decreased the accumulation of versican core protein, suggesting that transient treating of Chsy1-siRNA may be an alternative and an effective strategy to promote injured peripheral nerve regeneration.
... Upregulation of genes encoding CSPGs such as versican was also observed in the GBM microenvironment compared with normal tissues. Other studies, like ours, have shown higher levels of CSPGs in GBM tissues than in normal brains (37,38). ...
Unlabelled:
Glioblastomas (GBM) are aggressive brain tumors with extensive intratumoral heterogeneity that contributes to treatment resistance. Spatial characterization of GBMs could provide insights into the role of the brain tumor microenvironment in regulating intratumoral heterogeneity. Here, we performed spatial transcriptomic and single-cell analyses of the mouse and human GBM microenvironment to dissect the impact of distinct anatomical regions of brains on GBM. In a syngeneic GBM mouse model, spatial transcriptomics revealed that numerous extracellular matrix (ECM) molecules, including biglycan, were elevated in areas infiltrated with brain tumor-initiating cells (BTIC). Single-cell RNA sequencing and single-cell assay for transposase-accessible chromatin using sequencing showed that ECM molecules were differentially expressed by GBM cells based on their differentiation and cellular programming phenotypes. Exogeneous biglycan or overexpression of biglycan resulted in a higher proliferation rate of BTICs, which was associated mechanistically with low-density lipoprotein receptor-related protein 6 (LRP6) binding and activation of the Wnt/β-catenin pathway. Biglycan-overexpressing BTICs developed into larger tumors and displayed mesenchymal phenotypes when implanted intracranially in mice. This study points to the spatial heterogeneity of ECM molecules in GBM and suggests that the biglycan-LRP6 axis could be a therapeutic target to curb tumor growth.
Significance:
Characterization of the spatial heterogeneity of glioblastoma identifies regulators of brain tumor-initiating cells and tumor growth that could serve as candidates for therapeutic interventions to improve the prognosis of patients.
... Chondroitin sulfate A (CSA) is a biocompatible and biodegradable glycosaminoglycan that can bind to CD44 receptors, which are overexpressed on the surface of various tumor cells, including osteosarcoma [9][10][11][12]. As the CSA-CD44 interaction triggers endocytosis, this mechanism can be exploited to introduce cargo molecules (i.e., adsorbed or conjugated to CSA) into tumor cells, regardless of their hydrophilicity [9]. ...
Bone malignancy features a mineralized extracellular matrix primarily composed of hydroxyapatite, which interferes with the distribution and activity of antineoplastic agents. Herein, we report bone tumor-homing polymeric nanotherapeutics consisting of alendronate-decorated chondroitin sulfate A-graft-poly(lactide-co-glycolide) and doxorubicin (DOX), named PLCSA-AD, which displayed a prolonged retention profile in the tumor microenvironment and augmented therapeutic efficacy via inhibition of the mevalonate pathway. PLCSA-AD exhibited a 1.72-fold lower IC50 value than free DOX and a higher affinity for hydroxyapatite than PLCSA in HOS/MNNG cell-based 2D bone tumor-mimicking models. The inhibition of the mevalonate pathway by PLCSA-AD in tumor cells was verified by investigating the cytosolic fraction of unprenylated proteins, where blank PLCSA-AD significantly increased the expression of cytosolic Ras and RhoA without changing their total cellular amounts. In a bone tumor-mimicking xenografted mouse model, AD-decorated nanotherapeutics significantly increased tumor accumulation (1.73-fold) compared with PLCSA, and higher adsorption to hydroxyapatites was observed in the histological analysis of the tumor. As a result, inhibition of the mevalonate pathway and improvement in tumor accumulation led to markedly enhanced therapeutic efficacy in vivo, suggesting that PLCSA-AD could be promising nanotherapeutics for bone tumor treatment.
... Upregulation of genes encoding CSPGs including versican and CSPG4 was also observed in the GBM microenvironment compared to normal tissues. Other studies, like ours, have shown higher levels of CSPGs in GBM tissues than in normal brains 26,27 . Also, expression of CSPGs and enzymes involved in chondroitin sulphate polymerization is elevated in the GBM microenvironment and corresponds with high tumor grade and poor survival [28][29][30] . ...
Glioblastomas (GBMs) are aggressive brain tumors with extensive intratumoral heterogeneity. Here, we used spatial transcriptomics and single-cell ATAC-seq to dissect the transcriptome of distinct anatomical regions of the tumor microenvironment. We identified numerous extracellular matrix (ECM) molecules including biglycan elevated in areas infiltrated with brain tumor-initiating cells (BTICs). Single-cell RNA sequencing showed that the ECM molecules were differentially expressed by cells including injury response versus developmental BTICs. Exogeneous biglycan or overexpression of biglycan resulted in a higher proliferation rate of BTICs, and this was associated mechanistically with LDL receptor-related protein 6 (LRP6) binding and activation of the Wnt/β-catenin pathway. Biglycan-overexpressing BTICs grew to a larger tumor mass when implanted intracranially in mice. This study points to the spatial heterogeneity of ECM molecules in the GBM microenvironment and suggests biglycan-LRP6 axis as a therapeutic target to curb GBM growth.
... Indeed, while research has shown that hyperinsulinemia mediates pathologic angiogenesis through the proliferative actions of the ERK MAPK pathway (42,43), chronically elevated levels of insulin may also trigger the expression of EPDR1 and potentially many others through the endoglin/ALK1/Smad1/5 signaling axis. For instance, chondroitin sulfate synthase 2 (44,45) and prostaglandin F2 receptor-associated protein (46)(47)(48) are two of the top hits ranked higher than EPDR1, whereas others, including osteopontin (49,50) and platelet-derived growth factor receptor beta (51,52), represent promising candidates ranked slightly lower based on several parameters including fold changes over negative control and unique peptide counts identified (Fig. S3B). Future studies could explore whether these protein factors can serve as unique vascular targets during pathologic angiogenesis such as in diabetic retinopathy (53)(54)(55)(56)(57). ...
Insulin signaling in blood vessels primarily functions to stimulate angiogenesis and maintain vascular homeostasis through the canonical PI3K and MAPK signaling pathways. However, angiogenesis is a complex process coordinated by multiple other signaling events. Here we report a distinct crosstalk between the insulin receptor (IR) and endoglin/activin receptor-like kinase 1 (ALK1), an endothelial cell (EC)-specific TGF-β receptor complex essential for angiogenesis. While the endoglin/ALK1 complex normally binds to TGF-β or bone morphogenetic protein 9 (BMP9) to promote gene regulation via transcription factors Smad1/5, we show that insulin drives IR oligomerization with endoglin/ALK1 at the cell surface to trigger rapid Smad1/5 activation. Through quantitative proteomic analysis, we identify ependymin-related protein 1 (EPDR1) as a major Smad1/5 gene target induced by insulin, but not by TGF-β or BMP9. We found endothelial EPDR1 expression is minimal at the basal state but is markedly enhanced upon prolonged insulin treatment to promote cell migration and formation of capillary tubules. Conversely, we demonstrate EPDR1 depletion strongly abrogates these angiogenic effects, indicating that EPDR1 is a crucial mediator of insulin-induced angiogenesis. Taken together, these results suggest important therapeutic implications for EPDR1 and the TGF-β pathways in pathologic angiogenesis during hyperinsulinemia and insulin resistance.
... The experiment was repeated three times. Meanwhile, many research reported that hyaluronic acid (HA) and chondroitin sulfate (CS) could bind specifically with CD44 receptor 26,27 . To examine the uptake of CS/BH NPs was mediated by CD-44 receptors, cells were pretreated with excess free CS (5 mg/mL) or HA (5 mg/mL) for 1 h, respectively, then incubated with CS/BH NPs. ...
The poor prognosis of triple negative breast cancer (TNBC) results from a lack of approved targeted therapies coupled with aggressive proliferation and metastasis, which is associated with high recurrence and short overall survival. Here we developed a strategy by employing tumor-targeted self-assembled nanoparticles to coordinately regulate BACH1 (BTB domain and CNC homology 1) and mitochondrial metabolism. The BACH1 inhibitor hemin and mitochondria function inhibitor berberine derivative (BD) were used to prepare nanoparticles (BH NPs) followed by the modification of chondroitin sulfate (CS) on the surface of BH NPs to achieve tumor targeting (CS/BH NPs). CS/BH NPs were found to be able to inhibit tumor migration and invasion by significantly decreasing the amounts of tumor cell metabolites, glycolysis and metastasis-associated proteins, which were related to the inhibition of BACH1 function. Meanwhile, decreased mitochondrial membrane potential, activated caspase 3/9 and increased ROS production demonstrated coordinated regulation of BACH1 and mitochondrial metabolism. In a xenograft mice model of breast cancer, CS/BH NPs significantly inhibited tumor growth and metastasis due to the synergetic effect of hemin and BD without showing obvious toxicities for major organs. In sum, the results of efficacy and safety experiments suggest potential clinical significance of the prepared self-assembled CS/BH nanoparticles for the treatment of TNBC.
