Periodic Reporting for period 1 - PhotoStem (Photoswitching molecules for the spatiotemporal control of cancer stem cells with light)
Periodo di rendicontazione: 2019-09-01 al 2021-08-31
Cancer treatment still represents a major challenge, with continuous increase in incidence, morbidity and relapse. It is now accepted that resistance to conventional chemotherapy is often caused by a small population of cells that can self-renew and differentiate into the cells that constitute the bulk of the tumour. These are generally known as cancer stem cells (CSCs). CSCs usually adopt a quiescent state, which is not affected by standard anti-proliferative chemotherapy; most current treatments are able to eliminate the bulk of the tumour mass, but leave CSCs unaffected. This is thought to be the main cause of resistance to cancer treatment and tumour regrowth. Since the CSC concept was first demonstrated, the field of CSCs has seen an enormous advance, and to date CSCs have been identified, isolated and characterised from various human cancers. However, despite growing knowledge on the existence of these cells, finding effective and safe drugs against them is challenging.
In order to achieve higher complete remission rates after cancer treatment, eliminating CSCs is essential. One of the challenges in finding suitable drugs is their high similarity to healthy stem cells, which makes it difficult to design selective drugs that do not cause deleterious side effects in healthy tissues.
The lack of selectivity of small molecule drugs, which often leads to side effects, is caused by the inability to control their biological activity in time and space; this remains a major issue in the care of cancer patients amongst others. Photopharmacology aims to use light as an external non-invasive element to control drug activity, allowing for the activation of drugs with high spatial and temporal precision. It is based on the design of small molecules that have either a photoswitchable (reversible) or photocaging (irreversible) moiety, which delivers the active molecule only during/upon illumination with a particular light wavelength and intensity.
Within PhotoStem, we proposed that the use of photopharmacology techniques could overcome the challenge of differentiating between cancer and healthy stem cells. The overall aim was to design drugs that change their structure with light, and which can eliminate CSCs only after illumination. In the future, by illuminating only the tumour area, we would achieve tumour eradication, while leaving healthy stem cells of other tissues unaffected.
More specifically, the objectives were to firstly establish a proof of concept by (1) designing photoswitchable inhibitors of a well-known target, (2) testing their effect in CSCs in vitro and (3) in zebrafish; and secondly to (4) design light-activable inhibitors of a less explored pathway.
After this two-year fellowship, we have been able to establish a proof of concept in vitro. We first showed that photoswitchable inhibitors of histone deacetylases can eliminate CSC-enriched cell lines only under light conditions. We then also showed that a photocaged autophagy inhibitor could eliminate the formation of CSC spheres only after illumination. This represents an in vitro proof of concept that CSCs can be controlled with light using both photoswitchable and photocleavable molecules.
In order to achieve higher complete remission rates after cancer treatment, eliminating CSCs is essential. One of the challenges in finding suitable drugs is their high similarity to healthy stem cells, which makes it difficult to design selective drugs that do not cause deleterious side effects in healthy tissues.
The lack of selectivity of small molecule drugs, which often leads to side effects, is caused by the inability to control their biological activity in time and space; this remains a major issue in the care of cancer patients amongst others. Photopharmacology aims to use light as an external non-invasive element to control drug activity, allowing for the activation of drugs with high spatial and temporal precision. It is based on the design of small molecules that have either a photoswitchable (reversible) or photocaging (irreversible) moiety, which delivers the active molecule only during/upon illumination with a particular light wavelength and intensity.
Within PhotoStem, we proposed that the use of photopharmacology techniques could overcome the challenge of differentiating between cancer and healthy stem cells. The overall aim was to design drugs that change their structure with light, and which can eliminate CSCs only after illumination. In the future, by illuminating only the tumour area, we would achieve tumour eradication, while leaving healthy stem cells of other tissues unaffected.
More specifically, the objectives were to firstly establish a proof of concept by (1) designing photoswitchable inhibitors of a well-known target, (2) testing their effect in CSCs in vitro and (3) in zebrafish; and secondly to (4) design light-activable inhibitors of a less explored pathway.
After this two-year fellowship, we have been able to establish a proof of concept in vitro. We first showed that photoswitchable inhibitors of histone deacetylases can eliminate CSC-enriched cell lines only under light conditions. We then also showed that a photocaged autophagy inhibitor could eliminate the formation of CSC spheres only after illumination. This represents an in vitro proof of concept that CSCs can be controlled with light using both photoswitchable and photocleavable molecules.
The first stage of the project involved preparing photoswitchable inhibitors of histone deacetylases (HDACis), which would have better properties than already reported examples. For instance, we aimed for compounds activable by visible light and with significant differences in activity between dark and light conditions in both enzyme and cellular assays. Different structural modifications were introduced, based on known standard inhibitors and reported crystal structures. A library of over 40 photoswitchable compounds was constructed, which had a range of different photochemical properties such as photoswitching wavelengths and half-lives. In the second stage of the project, the biological activity of these compounds was evaluated in assays that were optimised for use under light conditions – firstly recombinant enzyme inhibitory assays, and then viability assays with cancer cell lines enriched in CSCs. Overall, we identified HDACis that were able to decrease viability of these cells only under illumination with either purple or green light, but not in the dark.
In the third stage of the project, a screen of compounds with known targets highlighted autophagy inhibitors as promising molecules against CSCs. Therefore, caged versions of the autophagy inhibitor chloroquine were prepared. Different cage groups were trialled, and the optimal one was chosen based on the release rate and yield under illumination. This caged chloroquine was able to disrupt the ability of HT29 cells to form CSC spheres only after illumination with blue light, but not in the dark.
Some of these results were communicated to the research community through a poster presentation at the EFMC Young Medicinal Chemists’ Symposium, and will be shortly published in peer-reviewed journals. In terms of a more general audience, the idea of the project was also published in a special edition of the outreach newsletter of the host institution “CSIC Investigates” on cancer research, which was also advertised in local newspapers, and it was also pitched in the Falling Walls Lab competition. We have also organised workshops and talks for school students.
In the third stage of the project, a screen of compounds with known targets highlighted autophagy inhibitors as promising molecules against CSCs. Therefore, caged versions of the autophagy inhibitor chloroquine were prepared. Different cage groups were trialled, and the optimal one was chosen based on the release rate and yield under illumination. This caged chloroquine was able to disrupt the ability of HT29 cells to form CSC spheres only after illumination with blue light, but not in the dark.
Some of these results were communicated to the research community through a poster presentation at the EFMC Young Medicinal Chemists’ Symposium, and will be shortly published in peer-reviewed journals. In terms of a more general audience, the idea of the project was also published in a special edition of the outreach newsletter of the host institution “CSIC Investigates” on cancer research, which was also advertised in local newspapers, and it was also pitched in the Falling Walls Lab competition. We have also organised workshops and talks for school students.
Given the strong evidence on the important role of CSCs in resistance to cancer treatment and relapse, extensive work is being done to find drugs that effectively eliminate them from the tumour. However, identifying drugs that do not affect healthy stem cells is an unresolved challenge. PhotoStem proposed that light could be the missing element that provides selectivity between CSCs and healthy stem cells. At the end of the project, we have proven for the first time that CSCs can be eliminated under light control, which represents an in vitro proof of concept of this idea.
After this in vitro proof of concept, the next steps beyond PhotoStem will be to optimise the molecules for in vivo use, and test their effect in in vivo models. Some of these studies will probably involve using a combination of our light-activable molecules together with an already-approved anticancer agent that targets the bulk tumour population. In parallel, we will collaborate with an engineering partner to develop medical devices that enable light delivery in a highly precise and controlled manner.
Before this therapy becomes a reality for patients, it will be necessary to undertake preclinical and clinical studies with the optimised light-activable molecules. If successful, this could at last represent an effective strategy to achieve full eradication of the tumour mass, hence providing long-term complete remission of cancer patients.
After this in vitro proof of concept, the next steps beyond PhotoStem will be to optimise the molecules for in vivo use, and test their effect in in vivo models. Some of these studies will probably involve using a combination of our light-activable molecules together with an already-approved anticancer agent that targets the bulk tumour population. In parallel, we will collaborate with an engineering partner to develop medical devices that enable light delivery in a highly precise and controlled manner.
Before this therapy becomes a reality for patients, it will be necessary to undertake preclinical and clinical studies with the optimised light-activable molecules. If successful, this could at last represent an effective strategy to achieve full eradication of the tumour mass, hence providing long-term complete remission of cancer patients.