Effect of Chitosan on Alginate-Based Macroporous Hydrogels for the Capture of Glioblastoma Cancer Cells.

Glioblastoma multiforme is a type of brain cancer associated with a very low survival rate since a large number of cancer cells remain infiltrated in the brain despite the treatments currently available. This work presents a macroporous hydrogel trap, destined to be implanted in the surgical cavity following tumor resection and designed to attract and retain cancer cells, in order to eliminate them afterward with a lethal dose of stereotactic radiotherapy. The biocompatible hydrogel formulation comprises sodium alginate (SA) and chitosan (CHI) bearing complementary electrostatic charges and stabilizing the gels in saline and cell culture media, as compared to pristine SA gels. The highly controlled and interconnected porosity, characterized by X-ray microCT, yields mechanical properties comparable to those of brain tissues and allows F98 glioblastoma cells to penetrate the gels within the entire volume, as confirmed by fluorescence microscopy. The addition of a grafted -RGD peptide on SA, combined with CHI, significantly enhances the adhesion and retention of F98 cells within the gels. Overall, the best compromise between low proliferation and a high level of accumulation and retention of F98 cells was obtained with the hydrogel formulated with 1% SA and 0.2% CHI, without the -RGD adhesion peptide.

An alginate-based macroporous hydrogel matrix to trap cancer cells.

To overcome the radioresistance of glioblastoma (GBM) cells infiltrated in the brain, we propose to attract these cancer cells into a trap to which a lethal radiation dose can be delivered safely. Herein, we have prepared and characterized a sodium alginate-based macroporous hydrogel as a potential cancer cell trap. Microcomputed X-ray tomography shows that the hydrogel matrices comprise interconnected pores with an average diameter of 300 µm. The F98 GBM cells migrated in the pores and mainly accumulated in the center of the matrix. Depending on the number of cancer cells added, the grafting of RGD cell-adhesion peptides to the alginate resulted in a 4 to 10 times increase in the number of F98 cells (which overexpress the associated αvß3 and αvß5 binding integrins) retained in the matrix. Finally, a radiation dose of 25 Gy eliminated all F98 cells trapped in the matrix, without significantly altering the matrix mechanical properties.

Erratum: Gascon S.; et al. Characterization and Mathematical Modeling of Alginate/Chitosan-Based Nanoparticles Releasing the Chemokine CXCL12 to Attract Glioblastoma Cells. Pharmaceutics 2020, 12, 356.

The authors wish to make the following correction to this paper [...].

Characterization and Mathematical Modeling of Alginate/Chitosan-Based Nanoparticles Releasing the Chemokine CXCL12 to Attract Glioblastoma Cells.

Chitosan (Chit) currently used to prepare nanoparticles (NPs) for brain application can be complexed with negatively charged polymers such as alginate (Alg) to better entrap positively charged molecules such as CXCL12. A sustained CXCL12 gradient created by a delivery system can be used, as a therapeutic approach, to control the migration of cancerous cells infiltrated in peri-tumoral tissues similar to those of glioblastoma multiforme (GBM). For this purpose, we prepared Alg/Chit NPs entrapping CXCL12 and characterized them. We demonstrated that Alg/Chit NPs, with an average size of ~250 nm, entrapped CXCL12 with ~98% efficiency for initial mass loadings varying from 0.372 to 1.490 µg/mg NPs. The release kinetic profiles of CXCL12 were dependent on the initial mass loading, and the released chemokine from NPs after seven days reached 12.6%, 32.3%, and 59.9% of cumulative release for initial contents of 0.372, 0.744, and 1.490 µg CXCL12/mg NPs, respectively. Mathematical modeling of released kinetics showed a predominant diffusive process with strong interactions between Alg and CXCL12. The CXCL12-NPs were not toxic and did not promote F98 GBM cell proliferation, while the released CXCL12 kept its chemotaxis effect. Thus, we developed an efficient and tunable CXCL12 delivery system as a promising therapeutic strategy that aims to be injected into a hydrogel used to fill the cavity after surgical tumor resection. This system will be used to attract infiltrated GBM cells prior to their elimination by conventional treatment without affecting a large zone of healthy brain tissue.

Tumor Cell Invasion Induced by Radiation in Balb/C Mouse is Prevented by the Cox-2 Inhibitor NS-398.

Radiation stimulates the expression of inflammatory mediators known to increase cancer cell invasion. Therefore, it is important to determine whether anti-inflammatory drugs can prevent this adverse effect of radiation. Since cyclooxygenase-2 (COX-2) is a central player in the inflammatory response, we performed studies to determine whether the COX-2 inhibitor NS-398 can reduce the radiation enhancement of cancer cell invasion. Thighs of Balb/c mice treated with NS-398 were irradiated with either daily fractions of 7.5 Gy for five consecutive days or a single 30 Gy dose prior to subcutaneous injection of nonirradiated MC7-L1 mammary cancer cells. Five weeks later, tumor invasion, blood vessel permeability and interstitial volumes were assessed using magnetic resonance imaging (MRI). Matrix metalloproteinase-2 (MMP-2) was measured in tissues by zymography at 21 days postirradiation. Cancer cell invasion in the mouse thighs was increased by 12-fold after fractionated irradiations (5 × 7.5 Gy) and by 17-fold after a single 30 Gy dose of radiation. This stimulation of cancer cell invasion was accompanied by a significant increase in the interstitial volume and a higher level of the protease MMP-2. NS-398 treatment largely prevented the stimulation of cancer cell invasion, which was associated with a reduction in interstitial volume in the irradiated thighs and a complete suppression of MMP-2 stimulation. In conclusion, this animal model using MC7-L1 cells demonstrates that radiation-induced cancer cell invasion can be largely prevented with the COX-2 inhibitor NS-398.

Radiation-induced lung metastasis development is MT1-MMP-dependent in a triple-negative breast cancer mouse model.

BACKGROUND: The prognosis of triple-negative breast cancer (TNBC) is still difficult to establish. Some TNBC benefit from radiotherapy (RT) and are cured, while in other patients metastases appear during the first 3 years after treatment. In this study, an animal model of TNBC was used to determine whether the expression of the cell membrane protease MT1-MMP in cancer cells was associated with radiation-stimulated development of lung metastases. METHODS: Using invasion chambers, irradiated fibroblasts were used as chemoattractants to assess the invasiveness of TNBC D2A1 cell lines showing downregulated expression of MT1-MMP, which were compared with D2A1-wt (wild-type) and D2A1 shMT1-mock (empty vector) cell lines. In a mouse model, a mammary gland was irradiated followed by the implantation of the downregulated MT1-MMP D2A1, D2A1-wt or D2A1 shMT1-mock cell lines. Migration of D2A1 cells in the mammary gland, number of circulating tumour cells and development of lung metastases were assessed. RESULTS: The reduction of MT1-MMP expression decreased the invasiveness of D2A1 cells and blocked the radiation enhancement of cancer cell invasion. In BALB/c mice, irradiation of the mammary gland has stimulated the invasion of cancer cells, which was associated with a higher number of circulating tumour cells and of lung metastases. These adverse effects of radiation were prevented by downregulating the MT1-MMP. CONCLUSIONS: This study shows that the MT1-MMP is necessary for the radiation enhancement of lung metastasis development, and that its expression level and/or localisation could be evaluated as a biomarker for predicting the early recurrence observed in some TNBC patients.

Induction of interleukin-1ß by mouse mammary tumor irradiation promotes triple negative breast cancer cells invasion and metastasis development.

PURPOSE: Radiotherapy increases the level of inflammatory cytokines, some of which are known to promote metastasis. In a mouse model of triple negative breast cancer (TNBC), we determined whether irradiation of the mammary tumor increases the level of key cytokines and favors the development of lung metastases. MATERIALS AND METHODS: D2A1 TNBC cells were implanted in the mammary glands of a Balb/c mouse and then 7 days old tumors were irradiated (4 × 6 Gy). The cytokines IL-1ß, IL-4, IL-6, IL-10, IL-17 and MIP-2 were quantified in plasma before, midway and after irradiation. The effect of tumor irradiation on the invasion of cancer cells, the number of circulating tumor cells (CTC) and lung metastases were also measured. RESULTS: TNBC tumor irradiation significantly increased the plasma level of IL-1ß, which was associated with a greater number of CTC (3.5-fold) and lung metastases (2.3-fold), compared to sham-irradiated animals. Enhancement of D2A1 cell invasion in mammary gland was associated with an increase of the matrix metalloproteinases-2 and -9 activity (MMP-2, -9). The ability of IL-1ß to stimulate the invasiveness of irradiated D2A1 cells was confirmed by in vitro invasion chamber assays. CONCLUSION: Irradiation targeting a D2A1 tumor and its microenvironment increased the level of the inflammatory cytokine IL-1ß and was associated with the promotion of cancer cell invasion and lung metastasis development.

Stimulation of triple negative breast cancer cell migration and metastases formation is prevented by chloroquine in a pre-irradiated mouse model.

BACKGROUND: Some triple negative breast cancer (TNBC) patients are at higher risk of recurrence in the first three years after treatment. This rapid relapse has been suggested to be associated with inflammatory mediators induced by radiation in healthy tissues that stimulate cancer cell migration and metastasis formation. In this study, the ability of chloroquine (CQ) to inhibit radiation-stimulated development of metastasis was assessed. METHODS: The capacity of CQ to prevent radiation-enhancement of cancer cell invasion was assessed in vitro with the TNBC cell lines D2A1, 4T1 and MDA-MB-231 and the non-TNBC cell lines MC7-L1, and MCF-7. In Balb/c mice, a single mammary gland was irradiated with four daily doses of 6 Gy. After the last irradiation, irradiated and control mammary glands were implanted with D2A1 cells. Mice were treated with CQ (vehicle, 40 or 60 mg/kg) 3 h before each irradiation and then every 72 h for 3 weeks. Migration of D2A1 cells in the mammary gland, the number of circulating tumor cells and lung metastasis were quantified, and also the expression of some inflammatory mediators. RESULTS: Irradiated fibroblasts have increased the invasiveness of the TNBC cell lines only, a stimulation that was prevented by CQ. On the other hand, invasiveness of the non-TNBC cell lines, which was not enhanced by irradiated fibroblasts, was also not significantly modified by CQ. In Balb/c mice, treatment with CQ prevented the stimulation of D2A1 TNBC cell migration in the pre-irradiated mammary gland, and reduced the number of circulating tumor cells and lung metastases. This protective effect of CQ was associated with a reduced expression of the inflammatory mediators interleukin-1ß, interleukin-6, and cyclooxygenase-2, while the levels of matrix metalloproteinases-2 and -9 were not modified. CQ also promoted a blocking of autophagy. CONCLUSION: CQ prevented radiation-enhancement of TNBC cell invasion and reduced the number of lung metastases in a mouse model.

Infiltration of F98 glioma cells in Fischer rat brain is temporary stimulated by radiation.

PURPOSE: Irradiation of brain stimulates the expression of inflammatory mediators, some of which can modify the ability of cancer cells to infiltrate the brain. In the present study, the time window during which this stimulation occurs was determined. MATERIALS AND METHODS: Brain of Fischer rat was irradiated (15 Gy) and expression of pro-inflammatory mediators IL-1ß, IL-6 and TNF-α was measured from 4 h to 20 days post-irradiation. Level of the matrix metalloproteinase 2 (MMP-2) and prostaglandin E2 (PGE2) which can favor cancer cell infiltration were also measured. The F98 glioma cells were implanted either during (4 h post-irradiation) or after (10 days post-irradiation) the pro-inflammatory phase. Infiltration distance of F98 cells in brain parenchyma and the median survival time of the animals were determined. RESULTS: Expression of IL-1ß, IL-6 and TNF-α was significantly increased in the irradiated brains with a peak at 4 h post-irradiation. Implantation of F98 glioma cells 4 h post-irradiation reduced the median survival time of Fischer rats to 18 days, compared to 25 days when the F98 were implanted in non-irradiated brain. Irradiation of the brain increased the distance of infiltration of F98 cells and was associated with increased levels of MMP-2 and PGE2. Conversely, F98 cells implanted 10 days post-irradiation have infiltrated the brain over a shorter distance and the median survival time of rats was increased to 35 days. CONCLUSIONS: Cancer recurrence is frequently observed in GBM patients. A better understanding of the inflammatory response observed in irradiated brain could contribute to develop new therapeutic modalities to further increase the efficiency of radiotherapy.

Role of interleukin-1ß in radiation-enhancement of MDA-MB-231 breast cancer cell invasion.

The ability of radiation to increase the invasiveness of cancer cells is associated with the inflammatory response, which is induced in almost all irradiated patients. For breast cancer patients, elevated plasma levels of the inflammatory cytokine interleukin-1ß (IL1ß) persisted for a few weeks after completion of radiotherapy. The aim of this study was to determine whether IL1ß is involved in the enhancement of breast cancer cell invasion induced by radiation. The role of IL1ß was assessed with invasion chambers where irradiated fibroblasts were used as chemoattractant for the MDA-MB-231 breast cancer cells plated in the upper compartment. The ability of IL1ß to stimulate the expression of cyclooxygenase-2 (COX-2) and biosynthesis of the prostaglandin E2 (PGE2) in MDA-MB-231 cells were also determined. Our results show that radiation-enhancement of MDA-MB-231 cell invasion was prevented with an anti-IL1ß antibody. The production of IL1ß was increased in irradiated fibroblasts, while the invasiveness of the MDA-MB-231 cells not exposed to irradiated fibroblasts was favored by adding this cytokine. Furthermore, addition of the COX-2 inhibitor NS-398 prevented the stimulation of cancer cell invasion induced either by irradiated fibroblasts or IL1ß. We propose that the effect of IL1ß on the invasiveness of the MDA-MB-231 cells involves elevation of matrix metalloproteinase-9 (MMP-9) production, induction of COX-2 expression and PGE2 biosynthesis. In conclusion, this study supports the involvement of IL1ß in the radiation-enhancement of breast cancer cell invasion.

New enzyme-activated solubility-switchable contrast agent for magnetic resonance imaging: from synthesis to in vivo imaging.

We designed and synthesized a novel contrast agent (CA) to image the activity of matrix metalloproteinase-2 (MMP-2) in a tumor, noninvasively using magnetic resonance imaging (MRI). We exploited the concept of solubility-switchable CAs in the design of a protease-modulated CA (PCA), referred to as PCA2-switch. This PCA contains a paramagnetic gadolinium chelate (Gd-DOTA), which was attached to the N-terminus of a MMP-2 cleavable peptide sequence via a hydrophobic chain. The aqueous solubility of the CA depends on the presence of a polyethylene glycol chain (PEG) on the C-terminus of the peptide. Upon proteolytic cleavage of the peptide by MMP-2, the PEG chain is detached from the CA, which becomes less water soluble. This compound and control compounds were successfully tested in an animal model bearing two tumors with different levels of MMP-2 activity.

Novel solubility-switchable MRI agent allows the noninvasive detection of matrix metalloproteinase-2 activity in vivo in a mouse model.

A novel MRI proteinase-modulated contrast agent (PCA) was developed to detect the activity of the proinvasive enzyme matrix metalloproteinase-2 (MMP-2) in vivo. The PCA2-switch agent incorporates a solubility switch, where cleavage of a peptide substrate by MMP-2 decreases the water solubility of the agent. Evidence suggests that this leads to an accumulation of cleaved PCA2-switch in an MMP-2-positive, wild-type, MC7-L1 mammary carcinoma tumor in a Balb/c mouse model compared to a MC7-L1 MMP-2-knockdown tumor. When a scrambled peptide sequence is inserted into the agent (PCA2-scrambled), the in vitro cleavage efficiency of MMP-2 is markedly reduced. In vivo, PCA2-scrambled does not accumulate in the wild-type tumor and the pharmacokinetics is similar in both tumors. In conclusion, in vivo cleavage of PCA2-switch by MMP-2 results in a significant accumulation of the cleaved PCA2-switch in an MMP-2-positive tumor.

Invasiveness of breast cancer cells MDA-MB-231 through extracellular matrix is increased by the estradiol metabolite 4-hydroxyestradiol.

In malignant breast cancer, estrogen metabolism is altered, favoring the accumulation of hydroxyestradiols, which can generate free radicals. These reactive species can activate matrix metalloproteinases (MMPs), which in turn can hydrolyze the proteins of the extracellular matrix (ECM) that act as a barrier to tumor cell passage. The aim of this study was to determine whether reactive oxygen species generated by 4-hydroxyestradiol (4-OHE(2)) can activate MMP-2 and then enhance the invasiveness of breast cancer cells MDA-MB-231 in vitro. Enzymatic assay and gel zymography demonstrated that 4-OHE(2) at a concentration as low as 10(-8) M led to the conversion of proMMP-2 to active MMP-2. Activation of proMMP-2 by 4-OHE(2) was inhibited by the Cu,Zn-SOD supporting the involvement of the free radical superoxide anion (O(2)(*-)). Using invasion chambers coated with matrigel (artificial ECM), 4-OHE(2) (10(-8) M) enhanced the invasiveness of MDA-MB-231 breast cancer cells by 3-fold. The addition of Cu,Zn-SOD reduced the invasiveness of MDA-MB-231 cells by more than 2-fold, supporting the involvement of O(2)(*-) generated by 4-OHE(2). Addition of an MMP-2 inhibitor completely inhibited the enhancement of invasiveness induced by 4-OHE(2), which demonstrates the importance of activating MMP-2 by 4-OHE(2). On the other hand, estradiol, which does not have a catechol structure, did not generate free radicals, and it could not activate proMMP-2 or enhance the invasiveness of beast cancer cells. Although these data need to be confirmed in an animal model, this study suggests that the accumulation of 4-OHE(2) in breast tumors could enhance the invasiveness of breast cancer cells.

Activation of matrix metalloproteinase-2 and -9 by 2- and 4-hydroxyestradiol.

Breast cancer patients frequently develop metastases. This process requires the degradation of extracellular matrix proteins which act as a barrier to tumour cell passage. These proteins can be degraded by proteases, mainly the matrix metalloproteinases (MMPs). MMP-2 and -9 which are frequently detected in breast cancer tissues. ProMMPs are released from cancer cells, and their activation is considered to be a crucial step in metastases development. In breast cancer, estrogen metabolism is altered favouring the accumulation of 2- and 4-hydroxyestradiol (2- and 4-OHE(2)). These estradiol metabolites can generate free radicals. Since reactive species are known activators of proMMPs, this study was designed to determine if the free radicals generated by 2- and 4-OHE(2) can activate proMMP-2 and -9. Activation of MMPs by hydroxyestradiol was determined by monitoring the cleavage of a fluorogenic peptide and by zymography analysis. Both estradiol metabolites activated the MMP-2 and -9. 4-OHE(2) was a more potent activator than 2-OHE(2), which reflects its higher capacity to generate free radicals. ProMMPs activation was mainly mediated through O(2)*-, although the free radical HO* also activated the proMMPs but to a lesser extent. ProMMPs activation was not observed with estrogens that cannot generate free radicals, i.e. estradiol, estrone, 2- and 4-methoxyestradiol, and 16alpha-hydroxyestrone. These results demonstrate that 2- and 4-OHE(2) at a concentration as low as 10(-8)M can activate the proMMP-2 and -9 and might play an important role in the invasion of breast cancer cells.

Vitamin C augments lymphocyte glutathione in subjects with ascorbate deficiency.

BACKGROUND: Ascorbate and glutathione play central roles in the defense against free radicals and oxidants that are implicated in chronic diseases. OBJECTIVE: The objective was to determine the ability of vitamin C supplements to modulate the concentration of glutathione in human lymphocytes. DESIGN: The effect of vitamin C supplements was determined in a sequential study with time points before supplementation, after 13 wk of vitamin C supplements (500 or 1000 mg/d), and after 13 wk of matching placebo. The supplementation group was selected on the basis of low plasma ascorbate (<33 mmol/L) and consisted of 48 healthy men and women, smokers and nonsmokers, aged 25-64 y. Ascorbate and glutathione were measured in purified lymphocytes. RESULTS: At baseline, the mean (+/-SD) concentration of plasma ascorbate was 19.5 +/- 7.2 micro mol/L, 22.5 micro mol/L below the median of normal distribution. The ascorbate concentration in plasma was linearly associated with that in lymphocytes (r = 0.53, P < 0.001). On supplementation with vitamin C, lymphocyte ascorbate increased by 51% (from 16.7 +/- 4.9 to 25.3 +/- 6.9 nmol/mg protein; P < 0.001) and was accompanied by an increase of lymphocyte glutathione by 18% (from 22.5 +/- 4.5 to 26.6 +/- 6.5 nmol/mg protein; P < 0.001). After placebo, the ascorbate and glutathione concentrations fell to near baseline concentrations (17.1 +/- 5.4 and 23.5 +/- 6.4 nmol/mg protein, respectively). No significant interaction was observed for sex and smoking status. Finally, the changes in lymphocyte ascorbate after supplementation were strongly associated with changes in lymphocyte glutathione (r = 0.71, P < 0.001). The association suggests that every 1-mol change in ascorbate is accompanied by a change of approximately 0.5 mol in glutathione. CONCLUSION: Vitamin C supplements increase glutathione in human lymphocytes.