Neutrophils and Snail Orchestrate the Establishment of a Pro-tumor Microenvironment in Lung Cancer

Understanding the immune compartment of tumors facilitates the development of revolutionary new therapies. We used a Kras(G12D)-driven mouse model of lung cancer to establish an immune signature and identified a contribution of Gr1 + neutrophils to disease progression. Depletion experiments showed that Gr1 + cells (1) favor tumor growth, (2) reduce T cell homing and prevent successful antiPD1 immunotherapy, and (3) alter angiogenesis, leading to hypoxia and sustained Snail expression in lung cancer cells. In turn, Snail accelerated disease progression and increased intratumoral Cxcl2 secretion and neutrophil infiltration. Cxcl2 was produced mainly by neutrophils themselves in response to a factor secreted by Snail-expressing tumor cells. We therefore propose a vicious cycle encompassing neutrophils and Snail to maintain a deleterious tumor microenvironment.

The role of Snail in non-small cell lung cancer

Svenja Johanna Groeneveld

The epithelial-to-mesenchymal transition (EMT) is a developmental program frequently reactivated in cancer. It plays an important role in several aspects of tumor progression, particularly in the acquisition of invasive capacities facilitating metastasis. Beyond invasion, EMT endows cancer cells with additional characteristics essential for metastatic dissemination and has been extensively studied in breast and colorectal cancer. Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related deaths worldwide and the majority of NSCLC patients is diagnosed with metastatic disease. The expression of the key EMT transcription factor Snail is correlated with a poor prognosis in NSCLC patients, while it is unclear whether Snail contributes to disease progression by actually facilitating metastasis formation. During EMT, the metabolism of a cancer cell changes, likely to meet the environmental challenges faced during the metastatic process. In this line, we have previously reported (Masin et al, 2014) that the glucose transporter GLUT3 is upregulated during EMT in human NSCLC cell lines. Here, we demonstrate that the rate-limiting enzyme of the hexosamine biosynthesis pathway (HBP) correlates with GLUT3 in NSCLC. This pathway produces a substrate for O-linked protein GlcNAcylations important during EMT, as they â for example â stabilize the Snail protein. Glutamine-fructose-6-phosphate amidotransferase 2 (GFPT2) was specifically upregulated during EMT, while its isoenzyme GFPT1 was unaffected. Furthermore, our results points towards a putative regulation of GLUT3 expression by GFPT2, possibly involving STAT3 signaling. We thus provide evidence for a possible reactivation of a developmental metabolic program during EMT in NSCLC. Chronic inflammation is an important disease-promoting factor during lung tumorigenesis, emphasizing the role of the immune system in this cancer type. Overexpressing or silencing Snail in the immunocompetent autochthonous KrasLSL-G12D/+;p53fl/fl mouse model of lung adenocarcinoma uncovered a substantial influence of Snail on the tumor microenvironment. An extensive analysis of tumor histology, gene and protein expression and immune infiltration revealed that while Snail did not contribute to metastasis formation, it accelerated growth and malignant progression of the lung tumors, reducing the survival time of the mice. Snail decreased B lymphocyte, while enhancing neutrophil infiltration of the tumors. Intriguingly, through the secretion of a soluble factor, Snail induced a feed-forward loop of neutrophil recruitment via Cxcl2, produced by neutrophils themselves. In our recently published collaborative work (Faget, Groeneveld et al, 2017), we identified neutrophils as main contributors to disease progression. We furthermore provided evidence for a vicious cycle formed between Snail expressing cancer cells and neutrophils in lung tumors, as neutrophils were found to impair angiogenesis, resulting in hypoxia and enhanced Snail expression. In addition, we found that Snail repressed the Dlk1-Dio3 locus, containing the genomeâs largest miRNA cluster and recently implicated in NSCLC, in tumor-infiltrating immune cells via a paracrine mechanism.

EPFL2018

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

Targeting and Modulating Tumor-Associated Sites with Novel Nanoparticle-Based Immunotherapies

Laura Jeanbart

Cancer is a global disease and a leading cause of death worldwide. While surgery, radiotherapy and chemotherapy comprise the classical tools to eradicate tumors, they do not cure cancer, cannot prevent metastases, lead to side effects, and most importantly they do not instruct the patientʼs immune system to recognize and fight tumor cells. Since the discovery of the immune systemʼs implication in cancer, immunotherapies, which aim at using and educating the immune system to fight and reject cancer, have come to complement classical tumor-debulking methods. A major goal of immunotherapy is to induce or activate effector cytotoxic CD8+ T lymphocytes that can infiltrate and kill the tumor while overcoming immune suppressive mechanisms, which impede their efficacy. Moreover, cancer immunotherapies aim at inducing memory to tumor antigens to prevent relapse or metastases, which traditional therapies cannot do. Dendritic cell (DC) targeting and activation are key for inducing adaptive immunity. Our laboratory has developed synthetic nanoparticles (NPs) capable of draining through lymphatics to target skin-draining lymph nodes (LNs) and being phagocytosed by resident antigen-presenting cells (APCs) upon intradermal injection. We developed a reproducible method to conjugate tumor antigens to NPs and co-delivered them with CpG-conjugated NPs. We found that a NP vaccine composed of a single melanoma-derived peptide (TRP- 2180-188) led to dramatic tumor growth delay in melanoma and was more efficient than a NP vaccine containing several tumor antigens from whole tumor cell lysate (TL). These findings contradicted our hypothesis that immunization with TL might lead to a broader T cell response and hence improved therapeutic outcomes. Additionally, we found that the dose of CpG could modulate the magnitude of the therapeutic outcomes in both single epitope and multiple antigen based NP-vaccines. Considering the efficacy of the developed NP vaccines, we used them as a tool to ask a fundamental and extremely relevant question regarding the influence of vaccine administration site on clinical efficacy: is it therapeutically more beneficial to target a tumor- draining lymph node (tdLN), which is immune suppressed but tumor antigen-primed, or a non-tdLN, which is neither suppressed nor primed by the tumor? We found that targeting the tdLN with NP vaccines led to significantly enhanced therapeutic outcomes in two different tumor models over targeting a non-tdLN, and that efficacy relied on NP conjugation of antigen and adjuvant. This suggested that antigen drainage from the tumor might be available to APCs in the tdLN and we further hypothesized that therapies that lead to immunogenic tumor cell death might synergize with a tdLN-targeted adjuvant approach. While cytotoxic modalities, such as chemotherapy and radiotherapy, have traditionally been used for their ability to kill tumor cells, tumor cells shed antigens and debris upon cell death that drain to the tdLN. Consistent with our hypothesis, targeting an adjuvant to the tdLN enhanced therapeutic benefits of chemo- and radio- therapy, while targeting a non-tdLN did not. We hypothesized that targeting the tdLN with NP vaccines might switch the tdLN microenvironment from a suppressive to an immunogenic one. This suggested that direct targeting of active suppressive mechanisms in the tumor might further improve immunotherapy. We thus engineered a nano-sized micellar carrier loaded with 6-thioguanine (MC-6TG), a cytotoxic drug used in leukemia, and explored its use in targeting T cell- impairing myeloid-derived suppressor cells (MDSCs) in order to enhance the efficacy of therapeutic vaccines. MC-6TG entirely eradicated both Ly6c+ monocytic and Ly6g+ granulocytic MDSCs for up to 7 d in the blood, tumor and tdLN of tumor-bearing mice. Since MC-6TG also targeted certain subsets of macrophages and DCs, depleting MDSCs did not have an impact on the efficacy of a NP vaccine, which relies on APCs for efficacy. However, MC-6TG synergized with adoptively transferred CD8+ T cells, significantly delaying tumor growth and enhancing survival in a murine model of implantable melanoma. Overall, this thesis describes the design and implementation of a novel approach to immunotherapy: we engineered NP-based therapeutic vaccines, optimized their delivery route to enhance efficacy, and simultaneously tackled immune suppressive cells in the tumor microenvironment to allow optimal therapeutic outcomes. The technologies and methods developed in this work are particularly relevant to clinical oncology and have the potential to benefit cancer patients by improving cancer therapy efficacy.

EPFL2013