Rachel Bujold

Pre-irradiation of mouse mammary gland stimulates cancer cell migration and development of lung metastases

Background:In most patients with breast cancer, radiotherapy induces inflammation that is characterised by an increase of promigratory factors in healthy tissues surrounding the tumour. However, their role in the emergence of the migration phenotype and formation of metastases is still unclear.Methods:A single mammary gland of BALB/c mice was irradiated with four doses of 6 Gy given at a 24-h interval. After the last session of irradiation, treated and control mammary glands were either collected for quantification of promigratory and proinflammatory factors or were implanted with fluorescent ubiquitination-based cell cycle indicator (FUCCI)-expressing mouse mammary cancer D2A1 cells. The migration of cancer cells in the mammary glands was monitored by optical imaging. On day 21, mammary tumours and lungs were collected for histology analyses and the quantification of metastases.Results:Pre-irradiation of the mammary gland increased by 1.8-fold the migration of cancer cells, by 2-fold the quantity of circulating cancer cells and by 2.4-fold the number of lung metastases. These adverse effects were associated with the induction of interleukin-6 (IL-6) and cyclooxygenase-2 (COX-2).Conclusion:The emergence of the metastasis phenotype is believed to be associated with the accumulation of mutations in cancer cells. Our results suggest an alternative mechanism based on promigratory factors from irradiated mammary glands. In clinic, the efficiency of radiotherapy could be improved by anti-inflammatory agents that would prevent the stimulation of cancer cell migration induced by radiation.

Infiltration of glioma cells in brain parenchyma stimulated by radiation in the F98/Fischer rat model

Abstract Purpose: In the months following radiotherapy, a rapid recurrence of glioblastoma multiforme occurs in the periphery of the resection cavity. The aim of this study was to assess the contribution of irradiation of the brain in the infiltration profile of glioma cells. Material and methods: Using the F98/Fischer rat glioma model, we either irradiated the brain, the F98 cancer cells, or both to separately investigate the effects of radiation. Inflammatory cytokines and pro-infiltration molecules were measured in irradiated brain. Results: A stimulation of interleukin-1β and transforming growth factor β1 expression 4 h after brain irradiation supported induction of inflammation. Early elevated expression of phospholipase A2 was also measured and was followed by a stimulation of cyclooxygenase-2 from day 5 to 20 after irradiation. This resulted in a biphasic increase of prostaglandins E2 and D2 biosynthesis with maximum at 4 h and 15 days post-irradiation. An important enhancement of F98 cells infiltration was observed when brain was irradiated, which took place at the expense of the growth of the primary tumour and resulted in a decreased median survival of the Fischer rats. This stimulation of F98 cells infiltration was associated with the pro-infiltration molecule, matrix metalloproteinase-2. Conclusion: In the animal model proposed, we demonstrated that irradiation of brain increased the infiltration capacity of F98 cells to the brain, resulting in a reduction of media survival of rats bearing this tumour. This animal model has also allowed identifying inflammatory cytokines and pro-infiltration molecules induced by radiation that can be targeted to prevent this adverse effect of radiation.

Irradiation of normal mouse tissue increases the invasiveness of mammary cancer cells

Purpose: Treatment of breast tumours frequently involves irradiating the whole breast to reach malignant microfoci scattered throughout the breast. In this study, we determined whether irradiation of normal tissues could increase the invasiveness of breast cancer cells in a mouse model. Materials and methods: Non-irradiated MC7-L1 mouse mammary carcinoma cells were injected subcutaneously in irradiated and non-irradiated thighs of Balb/c mice. The invasion volume, tumour volume, blood vessel permeability and interstitial volumes were monitored by magnetic resonance imaging (MRI). Slices of normal tissue invaded by cancer cells were examined by histology. Activity of matrix metalloproteinase -2 and -9 (MMP -2 and -9) in healthy and irradiated tissues was determined, and the proliferation index of the invading cancer cells was evaluated. Results: Three weeks after irradiation, enhancement of MC7-L1 cells invasiveness in irradiated thighs was already detected by MRI. The tumour invasion volume continued to extend 28- to 37-fold compared to the non-irradiated implantation site for the following three weeks, and it was associated with an increase of MMP-2 and -9 activities in healthy tissues. The interstitial volume associated with invading cancer cells was significantly larger in the pre–irradiated sites; while the blood vessels permeability was not altered. Cancer cells invading the healthy tissues were proliferating at a lower rate compared to non-invading cancer cells. Conclusion: Implantation of non-irradiated mammary cancer cells in previously irradiated normal tissue enhances the invasive capacity of the mammary cancer cells and is associated with an increased activity of MMP-2 and -9 in the irradiated normal tissue.

Radiation-enhancement of MDA-MB-231 breast cancer cell invasion prevented by a cyclooxygenase-2 inhibitor.

Background:Recent evidences support that radiation can promote the invasion of cancer cells. As interactions between cancer cells and surrounding stromal cells can have an important role in tumour progression, we determined whether an irradiation to fibroblasts can enhance the invasiveness of breast cancer cells. The role of cyclooxygenase-2 (COX-2), an inflammatory enzyme frequently induced by radiotherapy, was investigated.Methods:Irradiated 3T3 fibroblasts were plated in the lower compartment of invasion chambers and used as chemoattractant for non-irradiated human breast cancer cell MDA-MB-231, which are oestrogen receptor negative (ER(−)) and the oestrogen receptor positive (ER(+)) MCF-7 cells. Stimulation of COX-2 expression in irradiated 3T3 cells was measured by a semi-quantitative qPCR and western blot. Capacity of the major product of COX-2, the prostaglandin E2 (PGE2), to stimulate the production of the matrix metalloproteinase-2 (MMP-2) and cancer cell invasion were assessed with a zymography gel and invasion chambers.Results:Irradiation (5 Gy) of 3T3 fibroblasts increased COX-2 expression and enhanced by 5.8-fold the invasiveness of non-irradiated MDA-MB-231 cells, while their migration was not modified. Addition of the COX-2 inhibitor NS-398 completely prevented radiation-enhancement of cancer cell invasion. Further supporting the potential role of COX-2, addition of PGE2 has increased cancer cell invasion and release of MMP-2 from the MDA-MB-231 cells. This effect of radiation was dependant on the expression of membrane type 1 (MT1)–MMP, which is required to activate the MMP-2, but was not associated with the ER status. Although irradiated fibroblasts stimulated the invasiveness of MDA-MB-231 ER(−) cells, no enhancement was measured with the ER(+) cell line MCF-7.Conclusions:Radiation-enhancement of breast cancer cell invasion induced by irradiated 3T3 fibroblasts is not dependant on the ER status, but rather the expression of MT1–MMP. This adverse effect of radiation can be prevented by a specific COX-2 inhibitor.

Severe dose inaccuracies caused by an oxygen-antioxidant imbalance in normoxic polymer gel dosimeters.

Two oxygen scavengers have been successfully tested to produce normoxic polymer gel dosimeters under normal atmospheric conditions. The first is ascorbic acid and the second is a chloride (also sulfate) salt of tetrakis (hydroxymethyl) phosphonium. These antioxidants, added to the dosimeter during gel preparation, chemically remove dissolved oxygen that otherwise inhibits propagation of the polymerization reaction during irradiation of the dosimeter. These gel dosimeters are radiosensitive after manufacture under normoxic conditions. However, we show herein that the accuracy of the dosimetric measurement is compromised due to chemical reactions of the antioxidant with radicals. In addition, we provide evidence that both antioxidant and oxygen act as radical scavengers that affect the amount of polymer formed in the gel dosimeter. This can result in important dose inaccuracies in both methacrylic acid-based and acrylamide-based normoxic dosimeter gels.

Cyclooxygenase-2 inhibitor prevents radiation-enhanced infiltration of F98 glioma cells in brain of Fischer rat

Abstract Purpose: Radiation induces a neuro-inflammation that is characterized by the expression of genes known to increase the invasion of cancer cells. In Fischer rats, brain irradiation increases the infiltration of cancer cells and reduced the median survival of the animals. In this study, we have determined whether these adverse effects of radiation can be prevented with the cyclooxygenase-2 (COX-2) inhibitor meloxicam. Materials and methods: Brain of Fischer rats treated or not with meloxicam were irradiated (15 Gy) and then implanted with the F98 glioma cells. The median survival of the animals, the infiltration of F98 cells, and the expression of inflammatory cytokines and pro-migration molecules were measured. Results: Meloxicam reduced by 75% the production of prostaglandin E2 (bioproduct of COX-2) in irradiated brains validating its anti-inflammatory effect. Median survival was increased to control levels by the treatment of meloxicam following brain irradiation. This protective effect was associated with a reduction of the infiltration of F98 cells in the brain, a complete inhibition of radiation-enhancement of matrix metalloproteinase-2, and a significant reduction of tumor necrosis factor α (TNF-α) and tumor growth factor β1 (TGF-β1) expression. Using invasion chambers, interleukin-1β (IL-1β) stimulated by 5-fold the invasiveness of F98 cells, but this stimulation was completely inhibited by meloxicam. This suggests that a cooperation between IL-1β and COX-2 are involved in radiation-enhancement of F98 cell invasion. Conclusions: Our results indicate the importance of reducing the inflammatory response of normal brain tissue following irradiation in an effort to extend median survival in F98 tumor-bearing rats.

Volume-dependent internal temperature increase within polymer gel dosimeters during irradiation

The increase in temperature within polymer gel dosimeters due to exothermal polymerization reactions was measured during irradiation inside two normoxic polymer gels (MAGAT and PAGAT) with different volumes (from 18 ml to 257 ml) at a dose rate of 3.0 and 0.5 Gy.min−1. The same samples were evaluated by MRI the day after irradiation. The temperature rise is clearly a function of the irradiated gel volume and for the MAGAT gel dosimeter it is almost twice that of the PAGAT dosimeter. Herein, we report on the experimental set-up, preliminary temperature measurements and the corresponding MRI results.

Investigating potential physicochemical errors in polymer gel dosimeters.

Measurement errors in polymer gel dosimetry can originate either during irradiation or scanning. One concern related to the exothermic nature of polymerization reaction was that the heat released in polymer gel dosimeters during irradiation modifies their dose response. In this paper, the effect of heat released from the exothermal polymerization reaction on the dose response of a number of dosimeters was studied. In addition, we investigated whether heat-generated geometric distortion existed in newly proposed gel dosimeters that contain highly thermoresponsive polymers. Our results suggest that despite a significant internal temperature increase in some gel compositions, their dose responses are not affected when oxygen is well expelled mechanically from the gel mixture. We also report on significant pre-irradiation instability in some recently developed polymer gel dosimeters but that geometric distortions were not observed. Data obtained by a set of small calibration vials are compared to those obtained from larger phantoms, and potential physicochemical causes of deviations between them are identified.

Preliminary studies on the role and reactions of tetrakis(hydroxymethyl)phosphonium chloride in polyacrylamide gel dosimeters

A major source of dosimetric inaccuracy in normoxic polymer gel dosimeters is local variations in the concentration of oxygen scavenger. Currently, a phosphorus compound, tetrakis(hydroxymethyl)phosphonium chloride (THPC), is the oxygen scavenger of choice in most polymer gel dosimetry studies. Reactions of THPC in a gel dosimeter are not limited to oxygen. It can possibly be consumed in reacting with gelling agent, water free-radicals and polymer radicals before, during and after irradiation, hence affecting the dose response of the dosimeter in several ways. These reactions are not fully known or understood. It is our hypothesis that THPC not only scavenges radical species but also modifies the morphology of the gelatin network and of the polymer, possibly by intervening in the polymerization of monomers. These hypotheses are investigated in an anoxic acrylamide-based gel dosimeter. Scanning electron microscopy results indicate gelatin pores decreasing from 70 to 40 µm and a very different radiation-induced polymer structure in samples containing THPC; Fourier-transform Raman spectroscopy shows a two-fold reduction in the dose constants of monomer consumption; however, a significant change in the relative dose constants of monomer consumption as a function of dose could not be detected.

Effect of the exothermal polymerization reaction on polymer gel dosimetric measurements

Discrepancies in polymer gel dosimetric measurements have been observed between containers of different sizes receiving the same radiation dose. We hypothesized that these deviations are caused by a change in the rate of polymerization due to internal heat increase in the gel containers resulting from the exothermic polymerization of monomers. Here, we test this hypothesis in a polyacrylamide gel dosimeter by recording the temperature in glass phantoms of different sizes during and after irradiation. The dose response of the samples was determined with magnetic resonance imaging. The difference of R2 values along the depth of the containers was below ±1%. We discuss that this small difference can be attributed to variations in the rate of gelatin cooling during manufacture rather than to the measured heat increase during irradiation.