Microfluidics-assisted multiplexed biomarker detection for in situ mapping of immune cells in tumor sections

Because of the close interaction between tumors and the immune system, immunotherapies are nowadays considered as the most promising treatment against cancer. In order to define the diagnosis and the subsequent therapy, crucial information about the immune cells at the tumor site is needed. Indeed, different types or activation status of cells may be indicative for specific and personalized treatments. Here, we present a quantitative method to identify ten different immuno-markers in the same tumor cut section, thereby saving precious samples and enabling correlative analysis on several cell families and their activation status in a tumor microenvironment context. We designed and fabricated a microfluidic chip with optimal thermomechanical and optical properties for fast delivery of reagents on tissue slides and for fully automatic imaging by integration with an optical microscope. The multiplexing capability of the system is enabled by an optimized cyclic immunofluorescence protocol, with which we demonstrated quantitative sequential immunostaining of up to ten biomarkers on the same tissue section. Furthermore, we developed high-quality image-processing algorithms to map each cell in the entire tissue. As proofof-concept analyses, we identified coexpression and colocalization patterns of biomarkers to classify the immune cells and their activation status. Thanks to the quantitativeness and the automation of both the experimental and analytical methods, we believe that this multiplexing approach will meet the increasing clinical need of personalized diagnostics and therapy in cancer pathology.

Targeting cancer stem cells by T cell engineering

Nicolas Pierre Desbaillets

The identification of cancer stem cells (CSCs) has fundamentally changed the understanding of tumor biology. CSCs drive tumor initiation and long term tumor growth, can cause relapse and are essential for metastatic colonization. These features place cancer stem cells in the focus for cancer therapy. Recently, great advances have been made in the field of cancer adoptive immunotherapy using engineered T cells. Combining both fields of cancer research to generate immune therapies directly targeting CSCs for increased efficacy and long-term anti-tumor effects would generate great hope for cancer patients. Herein, proof of concept is made that chimeric antigen receptor (CARs) T cells killing breast CSCs are capable of eliminating established metastases. Killing of the CSCs modifies the immune environment resulting in the loss of regulatory T cells and in the induction of an endogenous immune response directed against the tumor that is indispensable for treatment success. Antibody mediated ablation of the CAR T cells induced a rapid re-appearance of CSCs and re-establishment of an immunosuppressive environment in the tumor creating a need for continuous targeting of the CSCs. I demonstrate that endogenous T cell responses are less effective against CSCs than the rest of the tumor. Furthermore, proof is made across multiple tumor models, that activated immune cells and inflammation induce dedifferentiation of bulk tumor cells which regain stem-like phenotypes. This observation further emphasizes the importance of directly targeting the CSCs. Studying the interplay between CSCs and the immune system requires syngeneic, immune competent murine models. This greatly limits the transgenic markers that one can use to visualize, track and target CSCs. Herein, we developed different tolerant mouse models to study breast cancer stem cells in vivo. 4T1 breast adenocarcinoma cells were engineered to express hCEA or GFP-based model surface antigens under the control a stem cell specific promoter. These cells, injected into syngeneic model antigen-tolerant mice established lung metastases which were eliminated by CAR T cell therapy. To further validate this approach using endogenous CSC markers, a genetic cancer model (MMTV-PyMT) has been developed, where stem cell Identification is based on CD90 allelic differentiation. Overall, this work highlights an important interplay between CSCs and the immune system. My results suggest that a feedback loop may be created were immune attack induces CSCs, which are immune privileged and escape killing from endogenous T cells. Subsequently, CSCs induce and control an immunosuppressive environment leading to immune escape. This provides a strong rationale to develop CSC-targeted therapies to be administered in combination with immunotherapies.

EPFL2017

New roles of the lymphatic endothelium in modulating adaptive immunity: implications for immune tolerance and cancer immunotherapy

Lambert Charles François Potin

Lymphatic vessels have traditionally been considered as passive conduits for interstitial fluid drainage and for immune cell trafficking to lymph nodes. In the last two decades, many studies have challenged this classical dogma: lymphatic endothelial cells (LECs) were shown to actively interact with immune cells by secreting chemokines and cytokines, and presenting antigens to T cells. These newly described functions pinpoint a multi-faceted role of LECs in orchestrating the immune response through both passive and active mechanisms. Yet, precise mechanisms leading to antigen-specific fine-tuning of the T cell response remain unexplored. Moreover, although lymphatics have been shown to play important roles in cancer, the contributions of LECs in regulating antitumor immunity have been largely overlooked by the biomedical community. This doctoral thesis aimed at filling these gaps. First, in order to investigate the mechanisms and implications of LEC antigen presentation to CD4+ T cells, we engineered a mouse model where LECs lacked the ability to present antigens to CD4+ T cells. In these mice, antigen-specific responses to vaccination were increased when compared to wild-type controls. Moreover, we demonstrated that LECs were poor inducers of CD4+ T cell activation in vitro, and that they could dampen CD4+ T cell activation by dendritic cells. Taken together, these findings suggest that LECs participate to the peripheral immune tolerance of CD4+ T cells. Second, we investigated the immunological consequences of the development of lymphatic vessels, called lymphangiogenesis, in mouse melanoma. Upon induction of tumor lymphangiogenesis, we observed striking tumor enrichment with immune cells, which resulted in a higher sensitivity of those tumors to immunotherapy. We subsequently demonstrated that inducing lymphangiogenesis in a mouse model of cold tumors, which lack immune infiltrates and do not respond to immunotherapies, could restore their responsiveness to immunotherapy treatments. Third, to predict which cancer types were the most likely to replicate the findings we obtained in mouse melanoma, we mined The Cancer Genome Atlas database for gene expression. We identified a set of cancers, such as colon adenocarcinoma and cholangiocarcinoma, where high expression of the lymphangiogenic growth factor was associated with a strong T cell signature. This doctoral thesis unveiled new immunomodulatory functions of LECs by which antigen-specific immune tolerance is induced. Additionally, it opened new avenues to target lymphatics therapeutically in cancer to potentiate immunotherapies. We believe this work contributes to demonstrating the immunological importance of LECs, and hope that lymphatics will be considered as an important immunomodulatory player of the tumor microenvironment when developing novel immunotherapies.

EPFL2018

An autonomous microfluidic device for identification and analysis of individual clinically-relevant mammalian cells

Fabien Louis Claude Robert Jammes

Cancer represents one of the major public health challenges worldwide, representing more than 25% of all deaths in the European Union in 2014. It affects a large and growing part of the general population, with the National Cancer Institute (NCI) estimating that nearly 40% of men and women will be diagnosed with cancer at some point in their lifetimes.Cancer presents an enormous intrinsic complexity and diversity as well as a pronounced resistance to interventions. Current treatments are composed primarily of chemotherapy, surgery, radiotherapy and drug therapy. Each of these approaches has been met with restrained successes and limitations. One growing field of cancer treatments that is showing promising results is immunotherapy. An improved understanding of the cancer biology and microenvironment over the years, particularly the interactions between a tumor and the immune system, has demonstrated the capability of the host immune system to detect and eliminate malignant cells through the process of immune surveillance. However, by various mechanisms, malignant cells can stochastically "hide" from the immune system and transform into tumors. Immunotherapies intend to use the immune system to fight the tumor progression. Different kinds of immunotherapies exist: from immune checkpoint inhibitors (PD-1), to monoclonal antibody treatments and cancer vaccines. One interesting approach of immunotherapy is called adoptive cell transfer therapy (ACT), wherein immune cells, and more precisely, T cells, from the cancer-affected patient are extracted, allowing tumor-specific and effective T cells to be isolated, expanded, activated and re-injected into the patient's body. This type of treatment has already demonstrated great tumor clearance potential as well as the ability to induce long-term cancer immunity. However, one of the major challenges in this type of treatment is the recognition, isolation and expansion of high-affinity, effective T cells that are able to eliminate cancer cells. Several key advances in the field of genetics, sequencing, and personalized medicine have allowed for the identification of tumor-specific antigens, the unique cancer signature for a given patient and a specific tumor. But there is a growing need for novel tools and platforms enabling the screening, selection and isolation of rare, naturally-occurring, high-affinity, tumor-specific T cells. In this thesis we present a novel approach to study the affinity of CD8+ lymphocytes at the single-cell level using microfluidics technology. We demonstrate the ability of our platform to isolate single cells and evaluate their affinities towards a given antigen by using the reversible pMHC NTAmers technology developed by Julien Schmidt et al. at the Ludwig Institute for Cancer Research in Lausanne, Switzerland. The innovative platform developed here effectively alleviates some of the bottlenecks of current technologies, like flow cytometry, for affinity studies of lymphocytes at the single-cell level. This thesis represents the development from idea to proof-of-concept of this novel platform and we herein describe the origins and characterization of this technology as well as the first applications towards a personalized-medicine tool for clinical research uses.

EPFL2021