Ph. D. Project
Title:
Role of claudins in the development of triple-negative breast cancer brain metastasis.
Dates:
2022/10/01 - 2025/09/30
Supervisor(s):
Description:
Breast cancer metastasis in the brain is significantly higher in triple negative breast cancers (TNBCs), (30%) and there is a lack of novel and/or improved therapies for these patients that have a median overall survival of 6 month. The molecular mechanisms that drive brain metastasis formation are poorly understood. In 76% of TNBC, the expression of claudin 1, a major constituent of tight junctions, has been shown to be absent or strongly diminished. Clinical studies reported that a loss of claudin 1 expression correlates with increased malignant and metastasis potential associated with recurrence of disease in invasive breast carcinoma patients. We showed that the re-expression of claudin 1 was sufficient to inhibit cell migration and increase intercellular adhesion in TNBC cells. Moreover, it induced apoptotic cell death and sensitized TNBC cells to chemotherapy. Our data highlight the importance of claudin 1 as therapeutic target in TNBC. Currently, no data are available on the role of claudin 1 in brain metastasis. Recently, one study showed that claudin 1 could suppress the formation of melanoma brain micro-metastasis in vivo by interaction with endothelial claudin 5. Thus, we hypothesis that claudins may have a role in TNBC brain metastasis.
The aim of the Ph.D is to evaluate the involvement claudins in the formation of TNBC brain metastasis. The project will present 2 parts
1) Investigate the role of claudins in the intravasation (blood-blood barrier) and extravasation (blood-brain barrier).
2) Investigate if the metastatic micro-environment could impair the claudin expression in the endothelial cells.
Experimental design
1. The Ph.D student will select the most relevant claudins using clinical data bases such as Breast Cancer Gene-Expression Miner v4.8. Then, he will analyze their expression in various cell model by RT-qPCR, western blot and immunolocalization. For the project, he will use two TNBC "claudin 1-low" cell line, MDA-MB-231 and Hs578T, and one TNBC "claudin 1-high" as well as HUVEC for endothelial barrier and hCMEC model of human blood-brain barrier (BBB) function.
Next, the involvement of the relevant claudin will be investigate using siRNA interference or over-expression to evaluate:
the role of claudins on adhesion mechanisms between TNBC cells and endothelial cells. Florescent dye will allow to discriminate endothelial cells (blue) and TNBC (green). The TNBC adhesion on endothelial cells will be monitored using Image X press imaging system.
the intravasation and extravasation mechanism. The Ph.D will study the cell migration of green labeled TNBC cells through a monolayer of endothelial cells HUVEC and/or hCMEC.
Then, the Ph.D student will validate the previous data on organotypic culture of mouse brain slices. The culture was developed in the lab. It allowed to monitor the migration of metastatic cells dyed with fluorescent probe. Briefly, a spheroid of TNBC overexpressing the relevant claudin will be implanted in the cortex of the right hemisphere. Control spheroid will be implanted in the left hemisphere. Cell invasion in the parenchyma will be monitored by Image X press imaging system.
2- Several factor such as ANGPTL4 or TGFimpaired the tight junction integrity through the down-regulation of claudins and promotes brain metastasis. In the same way, we hypothesis that the secretome of the tumoral environment could modify the level of claudin.
First, the Ph.D student will develop a spheroid co-culture model, closer to tumor micro-environment between TNBC and stromal cells. Then, he will compare the secretome of co-cultured spheroids, monolayer TNBC cells, or control medium. The endothelial monolayer cells will be place in presence of conditioned media at several time points. He will analyze by RT-qPCR, WB or immunolocalization whether claudins expression are modified. The component of the secretome responsible of the tight junction impairment (altered claudin expression) will be seek by mass spectrometry LC-MALDI analysis.
In conclusion, this work will determine 1) if claudins plays a key role in TNBC adhesion to endothelial cells and inhibits the metastasis formation, 2) if tumoral micro-environment could impaired claudins expression and alter endothelial barrier.
In future direction, this project aims to develop novel target / and drug that inhibit brain metastasis.
This project is part of our shift towards integrating the project: "System, Ionizing Radiation, in Neuro-Oncology" in the next contract. We used our expertise on claudin 1 and TNBC applied to brain metastasis. To the best of our knowledge, no teams are working on this specific subject. Interestingly, Izraely et al., described a key role of claudin 1 in melanoma brain and pulmonary metastasis (Izraely et al., 2015).
The aim of the Ph.D is to evaluate the involvement claudins in the formation of TNBC brain metastasis. The project will present 2 parts
1) Investigate the role of claudins in the intravasation (blood-blood barrier) and extravasation (blood-brain barrier).
2) Investigate if the metastatic micro-environment could impair the claudin expression in the endothelial cells.
Experimental design
1. The Ph.D student will select the most relevant claudins using clinical data bases such as Breast Cancer Gene-Expression Miner v4.8. Then, he will analyze their expression in various cell model by RT-qPCR, western blot and immunolocalization. For the project, he will use two TNBC "claudin 1-low" cell line, MDA-MB-231 and Hs578T, and one TNBC "claudin 1-high" as well as HUVEC for endothelial barrier and hCMEC model of human blood-brain barrier (BBB) function.
Next, the involvement of the relevant claudin will be investigate using siRNA interference or over-expression to evaluate:
the role of claudins on adhesion mechanisms between TNBC cells and endothelial cells. Florescent dye will allow to discriminate endothelial cells (blue) and TNBC (green). The TNBC adhesion on endothelial cells will be monitored using Image X press imaging system.
the intravasation and extravasation mechanism. The Ph.D will study the cell migration of green labeled TNBC cells through a monolayer of endothelial cells HUVEC and/or hCMEC.
Then, the Ph.D student will validate the previous data on organotypic culture of mouse brain slices. The culture was developed in the lab. It allowed to monitor the migration of metastatic cells dyed with fluorescent probe. Briefly, a spheroid of TNBC overexpressing the relevant claudin will be implanted in the cortex of the right hemisphere. Control spheroid will be implanted in the left hemisphere. Cell invasion in the parenchyma will be monitored by Image X press imaging system.
2- Several factor such as ANGPTL4 or TGFimpaired the tight junction integrity through the down-regulation of claudins and promotes brain metastasis. In the same way, we hypothesis that the secretome of the tumoral environment could modify the level of claudin.
First, the Ph.D student will develop a spheroid co-culture model, closer to tumor micro-environment between TNBC and stromal cells. Then, he will compare the secretome of co-cultured spheroids, monolayer TNBC cells, or control medium. The endothelial monolayer cells will be place in presence of conditioned media at several time points. He will analyze by RT-qPCR, WB or immunolocalization whether claudins expression are modified. The component of the secretome responsible of the tight junction impairment (altered claudin expression) will be seek by mass spectrometry LC-MALDI analysis.
In conclusion, this work will determine 1) if claudins plays a key role in TNBC adhesion to endothelial cells and inhibits the metastasis formation, 2) if tumoral micro-environment could impaired claudins expression and alter endothelial barrier.
In future direction, this project aims to develop novel target / and drug that inhibit brain metastasis.
This project is part of our shift towards integrating the project: "System, Ionizing Radiation, in Neuro-Oncology" in the next contract. We used our expertise on claudin 1 and TNBC applied to brain metastasis. To the best of our knowledge, no teams are working on this specific subject. Interestingly, Izraely et al., described a key role of claudin 1 in melanoma brain and pulmonary metastasis (Izraely et al., 2015).
Keywords:
Breast cancer, Claudin 1, brain metastasis, tight junction, cell migration
Conditions:
3 years
CRAN laboratory, UMR CNRS 7039 Biosis departent, Université de Lorraine, Campus science, boulevard des aiguillettes, entrée 1B,9eme étage BP 70 239 54 506 Vandoeuvre-les -Nancy France
1680 euros per month.
The candidate should have a Master degree in cell biology from an internationally recognized university. High level of competency in cell and molecular biology is required. The laureate should have obtained his diploma with honor (M1 and M2) and his academic cursus should be very good in order to be able to postulate to a Ph.D. fellowship.
CRAN laboratory, UMR CNRS 7039 Biosis departent, Université de Lorraine, Campus science, boulevard des aiguillettes, entrée 1B,9eme étage BP 70 239 54 506 Vandoeuvre-les -Nancy France
1680 euros per month.
The candidate should have a Master degree in cell biology from an internationally recognized university. High level of competency in cell and molecular biology is required. The laureate should have obtained his diploma with honor (M1 and M2) and his academic cursus should be very good in order to be able to postulate to a Ph.D. fellowship.
Department(s):
| Biology, Signals and Systems in Cancer and Neuroscience |
Funds:
3 years
Publications:
