Engineering Lymphangiogenesis | EPFL Graph Search

Melanoma causes the vast majority of skin cancer deaths, and there is currently no conventional treatment available that is able to cure metastatic melanoma patients. However, a novel treatment modality has recently emerged: cancer immunotherapy. Cancer immunotherapy aims at raising potent, tumor-specific T cell responses that are able to recognize and eliminate cancerous cells. Despite the fact that for the first time in history some metastatic melanoma patients can be cured, it is currently unclear why a large fraction of patients does not respond to immunotherapy. Thus, the overarching goal of this thesis was to gain a better understanding of the molecular mechanisms dictating the outcome to immunotherapy. Pre-existing inflammation within the tumor microenvironment is associated with good clinical prognosis following immunotherapy. Because our lab has previously shown that tumor associated lymphatic vessels (LVs) can modulate tumor inflammation, we hypothesized that LVs might be involved in modulating the outcome of cancer immunotherapy. To explore this hypothesis, we characterized two mouse models that recapitulate primary human lymphangiogenic melanoma. Using these models, we found that tumor-associated lymphatics secrete CCL21 to actively attract naïve T cells into the tumor microenvironment. Interestingly, we found that antigen-specific immunotherapy induced activation of these naïve T cell infiltrates, which not only resulted in eradication of the primary melanoma tumors, but also conferred the mice with long-term protection against tumor re-challenge. We thus demonstrate that LVs increase the quantity and quality of tumor inflammation, thereby establishing a microenvironment that is able to potentiate antigen-specific immunotherapy. We then show that controlled release of VEGFC from injectable hydrogels leads to lymphangiogenesis in the mouse dermis. Similarly to what we found in lymphangiogenic tumors, engineered lymphangiogenic skin sites displayed increased expression of CCL21, and increased infiltrations of CD4+ and CD8+ T cells. Delivery of the model antigen ovalbumin into lymphangiogenic sites led to enhanced release of the effector cytokine interferon-¿ upon antigen-restimulation, suggesting that engineered lymphangiogenic sites could be exploited for therapeutic immunomodulation. Finally, to better understand mechanisms underlying successful cancer immunotherapy, we established a tool for the observation of anti-tumor immune responses in the context of the native tumor microenvironment. By combining existing methods in a novel way, we developed an intravital microscopy method based on immunofluorescence that allows simultaneous, high resolution and dynamic visualization of the tumor microenvironment including immune cells and extracellular matrix proteins. In conclusion, this thesis demonstrates that lymphatic endothelial cells (LECs) orchestrate immune cell recruitment into peripheral tissues by secretion of CCL21 chemokine, both in steady-state and disease. Our findings add more evidence to the hypothesis that LECs might play an underappreciated role in driving the formation of peripheral sites of immunomodulation. Our findings have translational potential, as immune cell infiltrates attracted by local lymphangiogenesis can be exploited for therapeutic immune regulation, such as to improving the efficacy of cancer immunotherapy.

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

Therapeutic Immunomodulation of the Lymphoid-like Tumor Environment Using Nanoparticulate Formulations

Iraklis Kourtis

Cancer is a leading cause of death worldwide. Understanding the underlying mechanisms that lead to tumor escape and evasion is pivotal for the design of new therapeutic applications. This thesis takes an immunobioengineering approach, and navigates through identifying the immunological parameters relevant in the tumor microenvironment, proposing and developing a strategy in response to address these parameters using nanosized drug and antigen carriers. It is widely accepted that changes in the immunological equilibriumof the tumor microenvironment are crucial for the progression, dissemination and metastasis of a developing tumor. Motivated by tumors' natural secretion of CCL21, a chemokine known for dendritic (DC) migration towards the lymphatics, and for attracting DC and T cells in the lymph node (LN), we perturbed the physiological secretion of CCL21 of B16-F10 melanomas, deriving both a knock-down (CCL21low) and an over-expressing (CCL21high) variant. Interestingly, in immunocompetent mice, growth in CCL21-secreting tumors had an advantage over the CCL21low sub-clones and was CCR7 dependent. CCL21low tumors harbored more antigen specific T cells, while CCL21-secreting, more T regulatory cells (Tregs), which also correlated with subsequent biochemical polarization. Moreover, CCL21-secreting tumors formed a reticular fibroblastic (FRC) network (ERTR7+gp38+CCL21+) reminiscent of the lymphoid tissue of the paracortex (T cell zone) within LNs. The lymphoidlike stroma was orchestrated by the recruitment of CCR7+ adult lymphoid tissue inducers (LTis), responsible for LN formation during embryogenesis, present only in the CCL21-secreting tumors. In mice lacking LTis (Rorc(γt)-deficient) control tumors displayed impaired growth, suggesting that tumor growth is stroma dependent. In addition, the stroma of control or CCL21high tumors was infiltrated by more myeloid suppressor cells (MDSCs) than in CCL21low variant. MDSCs are a heterogeneous population of immature myeloid precursor cells that repress T cell activation and antigen presentation in chronic inflammation and cancer, hampering the efficacy of anti-tumoral vaccinations. CCL21's ability to protect allografts was further demonstrated in non-syngeneic recipients and in ectopically CCL21 transduced βTC tumor models. By mimicking secondary lymphoid organs, tumors may hi-jack and modulate the immune response from immunogenic to tolerogenic to allow for growth and metastasis. Intrigued by the excessive stroma infiltration of MDSCs in CCL21-secreting tumors, we performed a detailed spatiotemporal biodistribution analysis of ultrasmall (sub 50 nm) nanoparticles (NPs) in naïve and tumor bearing mice. Intradermally administered NPs rapidly drained to the LNs and spleen and persistently targetedmajor phagocytic populations that play a key role in hosting an immune response in naïve and tumor bearing mice. Furthermore, tumor-induced myeloid compartments, mainlyMDSCs, were highly targeted naturally within the tumormicroenvironment and spleen (tumor macroenvironment) 12 h post administration, without the use of site specific ligand. Following the MDSCs' natural preference towards phagocytosing NPs, and taking advantage of their excessive reductive cytoplasm, we designed a macromolecular pro-drug formulation that bears a cytotoxic payload and can be released in highly reductive microenvironments. Specifically, we engineered a reducible delivery system of 6-tioguanine (TG) in various formulations. Remarkably, bothMDSC compartments were ablated, monocytic ∼20 fold (CD11b+Ly6c+), polymorphonuclear ∼100 fold (CD11b+Ly6g+), for at least 7 d after administration in tumor bearing mice. As the bioidstribution revealed that apart from MDSCs, other antigen presenting cells (APCs) have phagocytosed NPs, we observed only a ∼2 fold decrease in APCs, suggesting that the rest could be used for vaccination. Taking into account that cancer vaccines and cancer immunotherapy rely on the efficient antigen presentation and education of naïve T cells by professional APCs, we explored the ability of NPs to deliver antigens in the appropriate sub-cellular compartment for antigen presentation and activation of T cells. Using the model antigen ovalbumin, we illustrated that exogenous antigen coupled to NPs by reducible bonds were internalized by DCs in vivo and in vitro. The conjugated antigen was preferentially released from the NPs with a reducible formulation, and was successfully presented in the major histocompatibility complex (MHC) I and II, which lead to activation and proliferation of antigen specific CD8+ and CD4+ T cells in vivo, respectively. Taking together, the nanoparticle platformcould be used efficiently for both inducing cytotoxicity and invoking an immune response. From discovery of new suppressive niches to therapeutical applications, we envisioned a two prong strategy that combined I) a modulatory regime to upset the balance of theMDSC populations both within the tumor microenvoronent and systemically with II) a cancer vaccine with the potential to significantly improve anti-tumoral therapeutic applications. This thesis demonstrated that such an approach is both possible and probable.

EPFL2012

The interplay between the VEGF-C/VEGFR-3 axis and CCL21/CCR7 axis in tumor cell invasion

Amine Issa

There are two main routes for tumor spread and metastasis, the blood vasculature and the lymphatic system. A wide range of human carcinomas, including lung, colon and breast cancer, spread to distant sites via the lymphatic system. Preclinical and clinical studies have demonstrated a positive correlation between the incidence of lymph node metastasis and the secretion of vascular endothelial growth factor-C (VEGF-C) by tumor cells. Indeed, VEGF-C is critical for the formation of new lymphatic vessels in a process called lymphangiogenesis, suggesting this mechanism as the main "escape route" for tumor cells. However, many studies have failed to identify functional lymphatic vessels inside tumors, and much research has demonstrated that metastasis can occur in the absence of lymphangiogenesis. Furthermore, there is growing evidence that many tumor cells may express vascular endothelial growth factor receptor-3 (VEGFR-3), the primary receptor for VEGF-C, suggesting that autologous VEGFR-3 signaling loops may enhance the invasive and metastatic potential of tumor cells. Other than lymphangiogenic factors, the lymphatic homing chemokine receptor CCR7 is strongly correlated to lymph node metastasis, suggesting that tumor cells mimic dendritic cells in sensing and homing to lymphatic vessels and lymph nodes. Potential mechanisms underlying these observations have not been elucidated. Using several in vitro 3D models, we examined the interactions between tumor and lymphatic endothelial cells (LECs) to gain insight into the mechanistic roles of VEGF-C and CCR7-mediated tumor cell invasion towards lymphatics as well as elucidate their potential interplay. All the designed experimental setups utilized a 3D matrix for a more physiologically relevant chemokine and growth factor signaling environment. For example, we designed one culture model where tumor cells are seeded in one gel compartment and lymphatic endothelial cells in a horizontally adjacent one, allowing both cell types to establish contact through soluble mediators without being initially mixed while simultaneously allowing direct visualization of their interactions. We first demonstrated that receptor expression patterns by cells are strongly influenced by the extracellular matrix (ECM). Indeed, while we failed to detect tumor expression of VEGFR-3 in 2D conditions, we found both expression and phosphorylation of VEGFR-3 in three of four tumor cell lines tested. Furthermore, in the three VEGFR-3 positive cell lines, invasive capacity and chemoattraction towards lymphatics was demonstrated to be dependent on both VEGF-C secretion and CCR7 expression by the tumor cells. We found that VEGF-C could act in both an autocrine manner, by enhancing the migratory and invasive capacity of tumor cells, and in a paracrine manner where it enhances CCL21 secretion by LECs to promote the chemoinvasion of tumor cells via CCL21 secretion. The same mechanism was also tested in vivo by injecting VEGF-C into mouse skin: VEGF-C, but not saline nor VEGF-A, caused marked upregulation of CCL21 associated with lymphatic vessels. In this way, VEGF-C and CCR7 act synergistically to promote the invasive phenotype of tumor cells. Additionally, we uncovered a new and critical role for VEGFR-3 on mammary epithelial cells. We demonstrated that the receptor is involved in epithelial tubulogenesis in a collagen matrix, and furthermore, it helps to preserve the integrity of a mammary cell layer (since blocking the receptor leads to increased epithelial permeability). In conclusion, this work highlights the importance of the VEGFR-3/VEGF-C and CCR7/CCL21 axis in promoting tumor cell migration and invasion. It also shows that VEGFR-3 may play many roles in the process of tumorigenesis and metastasis.

EPFL2009