Tumor lymphangiogenesis promotes T cell infiltration and potentiates immunotherapy in melanoma

In melanoma, vascular endothelial growth factor-C (VEGF-C) expression and consequent lymphangiogenesis correlate with metastasis and poor prognosis. VEGF-C also promotes tumor immunosuppression, suggesting that lymphangiogenesis inhibitors may be clinically useful in combination with immunotherapy. We addressed this concept in mouse melanoma models with VEGF receptor-3 (VEGFR-3)-blocking antibodies and unexpectedly found that VEGF-C signaling enhanced rather than suppressed the response to immunotherapy. We further found that this effect was mediated by VEGF-C-induced CCL21 and tumor infiltration of naive T cells before immunotherapy because CCR7 blockade reversed the potentiating effects of VEGF-C. In human metastatic melanoma, gene expression of VEGF-C strongly correlated with CCL21 and T cell inflammation, and serum VEGF-C concentrations associated with both T cell activation and expansion after peptide vaccination and clinical response to checkpoint blockade. We propose that VEGF-C potentiates immunotherapy by attracting naive T cells, which are locally activated upon immunotherapy-induced tumor cell killing, and that serum VEGF-C may serve as a predictive biomarker for immunotherapy response.

Role of Activin-A signaling in melanoma

Olivier Andreas Dubey

Cutaneous melanomas arise from transformed skin melanocytes. While accounting for only 1% of skin malignancies, melanoma is responsible for the majority of skin cancer-related deaths. Owing to lifestyle changes and increased exposure to sunlight, melanoma occurrence is on the rise in developed countries. Identification of the molecular mechanisms driving melanoma formation led to the development of new classes of therapeutics which revolutionized the standard of care. Among these, small molecule inhibitors targeting the MAPK pathway and immune checkpoint inhibitors resulted in unprecedented clinical success. Nevertheless, many patients still do not benefit from these therapies. Activin-A, a secreted ligand of the TGF-Î² superfamily, was recently found to be overexpressed in melanoma. However, whether Activin-A signaling promotes melanoma progression or therapy resistance is unknown. In this thesis, the contributions of autocrine and paracrine Activin-A signaling to melanoma growth are analyzed using lentiviral expression in melanoma cells of a constitutively active mutant type I Activin receptor (caALK4), of secreted Activin-A (gain-of-function), or of a ligand trap (loss-of-function). Paradoxically, while forced ALK4 signaling in melanoma grafts restored tumor suppression by inhibiting tumor cell survival and proliferation, transgenic Activin-A accelerated tumor growth. Conversely, expression of a ligand trap comprising the extracellular domain of Activin receptor IIB fused to human Fc (AIIB-Fc) slowed melanoma growth. Importantly, in immunodeficient Rag1-/- mice, effects of paracrine Activin-A gain and loss-of-function were abrogated, suggesting that Activin-A promotes melanoma progression by inhibiting the adaptive anti-tumor response. Flow cytometry analysis of tumor immune infiltrates combined with antibody-mediated cell depletion experiments confirmed that Activin-A promotes melanoma progression by inhibiting CD8+ tumor-infiltrating lymphocytes (TILs). Furthermore, inhibition of endogenous Activin-A sensitized a treatment-resistant melanoma model to immune checkpoint inhibition by anti-PD1 and anti-CTLA4 antibodies. Activin-A therefore emerges as a promising target to improve immunotherapy outcome in melanoma.

EPFL2019

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

Gene expression analysis of metastases and IL-1R1 characterization in a NALP3 mouse model

Heidi Fournier

Tumor microenvironment contributes strongly to tumor evolution toward metastasis, a final stage in cancer progression. It provides essential clues to promote migration of cancer cells in metastatic sites. Moreover, inflammatory cells and immunomodulatory mediators present in the microenvironment are able to polarize the host immune response, thereby potentiating primary tumor development. The reciprocal and dynamic interactions between microenvironment and tumor cells orchestrate critical steps of evolution toward metastasis. To address the role of inflammation in tumor progression, a mouse model of downregulated inflammation was used: the NALP3 gene in these mice is knocked-out, causing a impaired processing of the two inflammatory cytokines Interleukin-1[beta] and IL-18 which are essential for the recruitment and activation of immune cells. In this case, a decrease in tumor growth and metastasis was observed in previous studies, leading us to investigate the molecular and cellular interactions of tumor and immune cells in inflammatory and non-inflammatory contexts. Gene expression analysis was performed on two steps of mouse tumor cells: the B16-F10 melanoma cells seem to be more dependent on the immune cells recruitment to metastasize in vivo, while the Lewis Lung Carcinomas cells showed a quite equivalent growth in both wild-type or NALP3 knock-out mice. Different techniques were used to do so, such as standard in vitro assays and mRNA expression analyses, and in vivo tests with extraction of tumoral mRNA by Laser Capture Microdissection. Gene expression analyses of tumor cells were performed to address the influence of the inflammatory background. Comparison of these two gene expression profiles may reveal genes related to the specific reactive inflammatory response surrounding the given metastases. Another point that was investigated was the importance of the IL-1R1, receptor of interleukin-1. Since it represents the counterpart of IL-1[beta] signaling, its expression by immune and tumor cells may correlate with degree of tumor growth and invasion. In vitro and in vivo assays allowed better understanding the importance of this receptor. While a downregulation of this receptor impairs proliferation of in vitro cell cultures, at the in vivo level, the number of metastases is decreased too. These results provide potential clues indicating the important role played by the inflammatory cells, and how they can support and promote tumor progression, in particular through the expression of NALP3. The adaptation mechanisms of tumor cells to their microenvironment were also assessed, opening different insights for targeting the tumor itself as well as the inflammatory microenvironment.

2011