Periodic Reporting for period 1 - MOTM (Metabolic optimisation of intra-tumoral T cell motility)
Berichtszeitraum: 2023-01-01 bis 2024-12-31
Immunotherapies have transformed cancer treatment. However, only a minority of patients with solid cancers responds to these approaches, partly due to the complex environment of human tumors. In human solid cancer, tumor cells frequently lie within tumor islets surrounded by a stroma composed of fibers and host-derived cells that prevent T cells from efficiently making contact with tumor cells for their killing. In order to improve current immunotherapies, it is crucial to understand the molecular and cellular mechanisms underlying T cell sequestration in the tumor stroma to design innovative strategies rendering T cell successful in entering tumor islets. Metabolism controls several T cell functions, and it also sustains cell migration by supplying energy through glycolysis or oxidative phosphorylation (OXPHOS). Although the role of cellular metabolism in regulating the migration and formation of metastasis of cancer cells is well known, we currently ignore how metabolism regulates T cell migration in complex environment. Few studies have so far addressed this point, but they mostly used 2D in vitro systems, which are not adapted to investigate the 3D motility of T cells in a complex tumor microenvironment. Also, due to the unavailability of nutrients, the accumulation of waste products, the acid pH and the oxygen tension, T cell metabolism is severely altered within the tumor microenvironment. However, whether and how these deregulations precisely impact on intratumoral T cell motility is unknown. Therefore, the main objectives of the project were (i) to determine how metabolism regulates T cell 3D migration in complex environment, (ii) to understand whether and how metabolic alterations in the tumor microenvironment may impact on T cell motility and (iii) to identify new strategies to improve T cell intratumoral motility in the context of current immunotherapy approaches against solid cancer.
WP1 - Understanding the metabolic requirements for T cell migration.
A new assay based on 3D collagen gel has been developed to study the impact of metabolism on T cell 3D motility. Metabolism was manipulated in migrating T cells through the use of specific drugs and the add/removal of specific nutrients. Results were confirmed using genetic approaches (Crispr/Cas9) and by deposing T cells onto viable tumor slices from preclinical tumor models (human cancer cells implanted into immunodeficient mice). To investigate the mechanisms by which specific metabolic pathways modulate T cell 3D motility, experiments using inhibitors or modulators of ATP and ROS production were performed.
Outcomes. Overall, the experiments performed in WP1 allowed us to identify the main metabolic pathways supporting T cell 3D motility in a solid tumor microenvironment as well as how mechanistically the motility is supported by metabolism.
WP2 - Determine how the metabolic alterations of the TME affect intra-tumoral T cell motility.
An innovative strategy to measure T cell intratumoral motility and metabolic parameters in viable/fixed tumor slices obtained from human lung cancer tumors has been developed to look for correlations between motility and metabolism in cancer patients. A histo-cytometry pipeline has been developed in collaboration with IMAG’IC facility at host institute to analyse the expression of multiple metabolic markers (expression of key proteins associated with oxidative and glycolytic metabolism) in T cells infiltrating tumor biopsies from non-small cell lung cancer (NSCLC) patients. In a second phase, the impact of several metabolites enriched or deprived in the tumor microenvironment on T cell motility has been evaluated using collagen gels and viable tumor slices.
Outcomes. We identified a specific metabolic signature in highly motile T cells within the tumor microenvironment of non-small cell lung cancer (NSCLC) patients. Also, we highlighted the impact of some metabolic alterations frequently observed in the tumor microenvironment on T cell motility.
WP3 - Targeting metabolism to improve T cell intra-tumoral motility and infiltration in vivo.
The aim of this WP was to identify novel strategies to improve metabolically the intratumoral motility of T cells for immunotherapy applications. Based on the results obtained in WP1-2, it was decided to test several strategies to manipulate the metabolism of CAR T cells during in vitro generation/expansion in order to identify strategies able to improve migration through metabolic reprogramming. In a second phase one of these strategies (rapamycin treatment) was selected and used to generate CAR T cells whose anti-tumor ability was evaluated using preclinical xenograft lung tumor models in mice.
Outcomes. We identified an innovative strategy based on the manipulation of CAR T cell metabolism during in vitro generation that proved to be effective in increasing the infiltration and intratumoral motility of CAR T cells in preclinical xenograft human lung tumor models in mice.
A new assay based on 3D collagen gel has been developed to study the impact of metabolism on T cell 3D motility. Metabolism was manipulated in migrating T cells through the use of specific drugs and the add/removal of specific nutrients. Results were confirmed using genetic approaches (Crispr/Cas9) and by deposing T cells onto viable tumor slices from preclinical tumor models (human cancer cells implanted into immunodeficient mice). To investigate the mechanisms by which specific metabolic pathways modulate T cell 3D motility, experiments using inhibitors or modulators of ATP and ROS production were performed.
Outcomes. Overall, the experiments performed in WP1 allowed us to identify the main metabolic pathways supporting T cell 3D motility in a solid tumor microenvironment as well as how mechanistically the motility is supported by metabolism.
WP2 - Determine how the metabolic alterations of the TME affect intra-tumoral T cell motility.
An innovative strategy to measure T cell intratumoral motility and metabolic parameters in viable/fixed tumor slices obtained from human lung cancer tumors has been developed to look for correlations between motility and metabolism in cancer patients. A histo-cytometry pipeline has been developed in collaboration with IMAG’IC facility at host institute to analyse the expression of multiple metabolic markers (expression of key proteins associated with oxidative and glycolytic metabolism) in T cells infiltrating tumor biopsies from non-small cell lung cancer (NSCLC) patients. In a second phase, the impact of several metabolites enriched or deprived in the tumor microenvironment on T cell motility has been evaluated using collagen gels and viable tumor slices.
Outcomes. We identified a specific metabolic signature in highly motile T cells within the tumor microenvironment of non-small cell lung cancer (NSCLC) patients. Also, we highlighted the impact of some metabolic alterations frequently observed in the tumor microenvironment on T cell motility.
WP3 - Targeting metabolism to improve T cell intra-tumoral motility and infiltration in vivo.
The aim of this WP was to identify novel strategies to improve metabolically the intratumoral motility of T cells for immunotherapy applications. Based on the results obtained in WP1-2, it was decided to test several strategies to manipulate the metabolism of CAR T cells during in vitro generation/expansion in order to identify strategies able to improve migration through metabolic reprogramming. In a second phase one of these strategies (rapamycin treatment) was selected and used to generate CAR T cells whose anti-tumor ability was evaluated using preclinical xenograft lung tumor models in mice.
Outcomes. We identified an innovative strategy based on the manipulation of CAR T cell metabolism during in vitro generation that proved to be effective in increasing the infiltration and intratumoral motility of CAR T cells in preclinical xenograft human lung tumor models in mice.
The results of the MOTM project have been published in an open-access article in the journal Nature Communications (doi: 10.1038/s41467-024-46377-7). Here summary of the results obtained:
-) we have identified the metabolic requirements of human T cells migrating in a 3D environment, with a special focus on the tumor microenvironment. Specifically, T cell 3D motility is supported by TCA cycle fueled by glucose and glutamine but not fatty acids (Figure 1 of the Nat Commun paper).
-) we demonstrated the relative minor role played by glycolysis compared with mitochondrial OXPHOS in supporting human T cell 3D motility (Figure 2 of the Nat Commun paper).
-) we showed that human T cell 3D motility and mitochondrial metabolism are positively correlated in different contexts both at intra- and inter-population levels, including CD8+ T cells infiltrating non-small cell lung cancer (Figure 3 of the Nat Commun paper).
-) mechanistically, we identified both mitochondrial ATP and mitochondria-derived ROS (especially those produced by ETC complex II) as critical molecules produced by mitochondrial metabolism to sustain positively human T cell 3D migration (Figure 4 of the Nat Commun paper).
-) we have identified different strategies able to improve T cell 3D motility through a metabolic reprogramming (Figure 5 and Figure 8 of the Nat Commun paper). Among them, we further showed that treatment with rapamycin improves not only human T cell motility within a tumor microenvironment (Figure 6 of the Nat Commun paper) but also the infiltration of human CAR T cells into two preclinical xenograft human lung tumor models implanted in mice (Figure 7 of the Nat Commun paper), thus improving tumor control.
Overall, we have defined for the first time the metabolic requirements of T cells migrating within a solid tumor mass and we showed how metabolism in the tumor microenvironment may influence the motility of tumor-infiltrating T cells. Our study advances the field by providing two major advances:
-) First, by showing how mitochondrial metabolism supports the ability of T cells to migrate and infiltrate tumor islets, we provide an additional explanation for understanding the efficacy of antitumor strategies targeting mitochondria in T cells beyond the well-established effect on T cell persistence (which is the only explanation currently proposed in the field).
-) Second, from a clinical perspective, we have developed a simple in vitro pharmacological approach that allows the generation of CAR T cells with improved ability to infiltrate human solid tumors and with strong translational potential since it could be easily implemented in current CAR T cell production protocols. To our knowledge, this is the first immunometabolism approach against solid cancers focused on improving intratumoral CAR T cell motility.
These results of the project will be used by the MSCA fellow to apply for new research grants in the next years in order to continue the research on the topic of cancer immunotherapy and further extend the impact of the project for biomedical application in the immunotherapy field. The scientific impact of the MOTM action has been fully achieved, as the main questions posed in the project have been answered. No direct application in economic, societal, or industrial production or processes fields is envisaged now. However, the results of the MOTM action represent the basis for further scientific research that may lead to the setup of new immunotherapy approaches to fight against solid cancer and, therefore, may have an impact at industrial and well-being fields in the mid-term future. In line with this discussion is ongoing with clinicians at host institute Hospital center (Cochin) to implement the application of the identified strategies in clinical trials.
-) we have identified the metabolic requirements of human T cells migrating in a 3D environment, with a special focus on the tumor microenvironment. Specifically, T cell 3D motility is supported by TCA cycle fueled by glucose and glutamine but not fatty acids (Figure 1 of the Nat Commun paper).
-) we demonstrated the relative minor role played by glycolysis compared with mitochondrial OXPHOS in supporting human T cell 3D motility (Figure 2 of the Nat Commun paper).
-) we showed that human T cell 3D motility and mitochondrial metabolism are positively correlated in different contexts both at intra- and inter-population levels, including CD8+ T cells infiltrating non-small cell lung cancer (Figure 3 of the Nat Commun paper).
-) mechanistically, we identified both mitochondrial ATP and mitochondria-derived ROS (especially those produced by ETC complex II) as critical molecules produced by mitochondrial metabolism to sustain positively human T cell 3D migration (Figure 4 of the Nat Commun paper).
-) we have identified different strategies able to improve T cell 3D motility through a metabolic reprogramming (Figure 5 and Figure 8 of the Nat Commun paper). Among them, we further showed that treatment with rapamycin improves not only human T cell motility within a tumor microenvironment (Figure 6 of the Nat Commun paper) but also the infiltration of human CAR T cells into two preclinical xenograft human lung tumor models implanted in mice (Figure 7 of the Nat Commun paper), thus improving tumor control.
Overall, we have defined for the first time the metabolic requirements of T cells migrating within a solid tumor mass and we showed how metabolism in the tumor microenvironment may influence the motility of tumor-infiltrating T cells. Our study advances the field by providing two major advances:
-) First, by showing how mitochondrial metabolism supports the ability of T cells to migrate and infiltrate tumor islets, we provide an additional explanation for understanding the efficacy of antitumor strategies targeting mitochondria in T cells beyond the well-established effect on T cell persistence (which is the only explanation currently proposed in the field).
-) Second, from a clinical perspective, we have developed a simple in vitro pharmacological approach that allows the generation of CAR T cells with improved ability to infiltrate human solid tumors and with strong translational potential since it could be easily implemented in current CAR T cell production protocols. To our knowledge, this is the first immunometabolism approach against solid cancers focused on improving intratumoral CAR T cell motility.
These results of the project will be used by the MSCA fellow to apply for new research grants in the next years in order to continue the research on the topic of cancer immunotherapy and further extend the impact of the project for biomedical application in the immunotherapy field. The scientific impact of the MOTM action has been fully achieved, as the main questions posed in the project have been answered. No direct application in economic, societal, or industrial production or processes fields is envisaged now. However, the results of the MOTM action represent the basis for further scientific research that may lead to the setup of new immunotherapy approaches to fight against solid cancer and, therefore, may have an impact at industrial and well-being fields in the mid-term future. In line with this discussion is ongoing with clinicians at host institute Hospital center (Cochin) to implement the application of the identified strategies in clinical trials.