Targeting cancer-associated fibroblasts to enhance the radiation-induced anti-tumor immune response
PURPOSE: The objective of this project is to develop and test methods that selectively deplete or increase cancer-associated fibroblasts (CAFs) on solid tumors, and to use this approach to 1) study the role of CAFs in the response of tumors to radiotherapy and 2) enhance radiation-induced anti-tumor immune responses.
VALUE for SOCIETY: Radiation therapy (RT) has emerged as an appealing partner for cancer immunotherapies, given its dual cytotoxic and immunomodulatory properties. CAFs are cells residing in tumors, they play important roles in tumor development and participate actively in tumor immunoregulation, however, their influence on immune responses in the context of radiotherapy is still poorly understood. In this project, we hypothesize that CAF selective targeting could have advantageous effects on radiotherapy and immunotherapy approaches. The development of novel approaches that will permit better stratification of patients together with the implementation of combinatory treatment strategies focusing on enhancing response rates, will represent a big leap in the field of cancer therapy with the potential for boundless benefit to society.
METHODS:
We will use subcutaneous allograft tumor models, created by injection of transplantable tumor cells in the flank of syngeneic mice. Animals will be subjected to selective depletion of CAFs using three different approaches: 1) local tumor injections of the chemical agent TRANILAST (antifibrotic agent); ip. administration of 2) PIRFENIDONE (antifibrotic agent), or 3) CALCIPOTRIOL (Vitamin D). To increase CAFs abundance, tumor cells will be co-injected with activated mouse fibroblasts. In experiments involving radiotherapy, individual tumors will be exposed to ionizing radiation by the use of an image-guided animal irradiator. Successful CAF depletion will be confirmed by the CAF-specific PET radiotracer FAPI-74. Cytotoxic lymphocyte infiltration will be studied by PET imaging using CD8+ specific radiotracer.
PAIN & DISCOMFORT: The tumor models, radiation treatment protocols, and imaging methods that we will use are considered to cause minor discomfort/pain in the animals. Animals will be sedated, kept warm, and closely monitored for vital parameters during imaging and treatment procedures. Animals will be humanely euthanized at the end of the protocol.
NUMBER of ANIMALS: Pilot experiments are planned to test the efficacy of CAF depletion approaches before the execution of large experiments. We are applying for a total of 328 C57Bl/6 and 192 129Sv/Ev. To increase the robustness and reproducibility of results, identical experiments will be reproduced in two different tumor models, comprising two different mouse strains (C57Bl/6 and BALB/c).
ETHICS: Reduction: the number of pilot experiments will be reduced to the minimum since experimental protocols related to the establishment of tumor models and radiation protocols will be in place. Refinement: Pilot studies with a small number of animals will be carried out to establish CAF-depletion approaches, test the efficacy of the methods, and monitor potential toxicities. Replacement: Large amount of data is being collected from in vitro experiments, however, the use of in vivo tumor models is unavoidable as radiotherapy treatment responses and therapy-mediated immune activation can only be study using whole organisms.
VALUE for SOCIETY: Radiation therapy (RT) has emerged as an appealing partner for cancer immunotherapies, given its dual cytotoxic and immunomodulatory properties. CAFs are cells residing in tumors, they play important roles in tumor development and participate actively in tumor immunoregulation, however, their influence on immune responses in the context of radiotherapy is still poorly understood. In this project, we hypothesize that CAF selective targeting could have advantageous effects on radiotherapy and immunotherapy approaches. The development of novel approaches that will permit better stratification of patients together with the implementation of combinatory treatment strategies focusing on enhancing response rates, will represent a big leap in the field of cancer therapy with the potential for boundless benefit to society.
METHODS:
We will use subcutaneous allograft tumor models, created by injection of transplantable tumor cells in the flank of syngeneic mice. Animals will be subjected to selective depletion of CAFs using three different approaches: 1) local tumor injections of the chemical agent TRANILAST (antifibrotic agent); ip. administration of 2) PIRFENIDONE (antifibrotic agent), or 3) CALCIPOTRIOL (Vitamin D). To increase CAFs abundance, tumor cells will be co-injected with activated mouse fibroblasts. In experiments involving radiotherapy, individual tumors will be exposed to ionizing radiation by the use of an image-guided animal irradiator. Successful CAF depletion will be confirmed by the CAF-specific PET radiotracer FAPI-74. Cytotoxic lymphocyte infiltration will be studied by PET imaging using CD8+ specific radiotracer.
PAIN & DISCOMFORT: The tumor models, radiation treatment protocols, and imaging methods that we will use are considered to cause minor discomfort/pain in the animals. Animals will be sedated, kept warm, and closely monitored for vital parameters during imaging and treatment procedures. Animals will be humanely euthanized at the end of the protocol.
NUMBER of ANIMALS: Pilot experiments are planned to test the efficacy of CAF depletion approaches before the execution of large experiments. We are applying for a total of 328 C57Bl/6 and 192 129Sv/Ev. To increase the robustness and reproducibility of results, identical experiments will be reproduced in two different tumor models, comprising two different mouse strains (C57Bl/6 and BALB/c).
ETHICS: Reduction: the number of pilot experiments will be reduced to the minimum since experimental protocols related to the establishment of tumor models and radiation protocols will be in place. Refinement: Pilot studies with a small number of animals will be carried out to establish CAF-depletion approaches, test the efficacy of the methods, and monitor potential toxicities. Replacement: Large amount of data is being collected from in vitro experiments, however, the use of in vivo tumor models is unavoidable as radiotherapy treatment responses and therapy-mediated immune activation can only be study using whole organisms.