URPP Fellow: Nicolás Núñez

Conventional cancer therapies rely on targeting the malignant cells through surgery, radiation and/or chemotherapy. At this point in time, it is unlikely that such strategies will provide additional therapeutic benefit and in particular treatment resistant solid-tissue cancers require novel approaches. Immune subversion by cancers is by now well documented and accepted and reversal of this process as well as activation of anti-cancer immunity is likely the most promising strategy to combat cancer in the years to come. The success of check point blockers in metastatic refractory melanoma highlights the potential of cancer immunotherapy to not only halt cancer progression, but also to cure established malignancies. One puzzling aspect is the relatively small percentage of patients, which show long-term progression free survival. It appears that even strong anti-tumor immune responses fail to penetrate the tumors.
Cancers not only contain transformed cells, but also actively shape their microenvironment, which is made up of non-transformed stromal, endothelial and immune cells. The impact of such cells on tumor progression, the formation of metastases, and the response to standard or immune therapies is largely unknown but of great interest and an emerging field of research. In the context of immunotherapy for example, between 20 and 40% of patients respond to checkpoint blockade, implicating that a large percentage of patients still do not respond. Suboptimal or absent responsiveness in those patients is also reported for other treatments, including standard therapies. We propose that the tumor microenvironment influences the clinical response to treatment. In particular, we think that the tumor-associated innate leukocyte compartment, which makes up the majority of the non-malignant cells in a tumor, impacts on the adaptive anti-tumor response in most patients and as such, sets the course for response to therapy.
Our previous results show that before commencing therapy, a strong predictor of progression-free and overall survival in response to anti-PD-1 immunotherapy was the frequency of CD14+CD16-HLA-DRhi monocytes. We expanded our findings in a new cohort of patients with melanoma receiving combinatorial treatment (anti-PD1 and anti-CTLA-4).  Our results show that during therapy, a clear response to immunotherapy in the T cell compartment was observed. More precisely, before starting the combinatorial treatment we observed in responders compared to non-responders higher frequency of CD8 T cells, with a signature of IFN+, TNF+, Gzmb+.  Furthermore, after 12 weeks of combinatorial treatment we observed an expansion of CD4+FOXP3+ T cells population only in responders.
Here, we wish to delve deeper analyze the immune compartment by multi-dimensional and unbiased using CyTOF, FACSymphony and bioinformatics, and making use of a unique genetically characterized patient-derived tumor samples and live tumor cells deposited in our URPP tumor cell biobank.

Aims

1. Since we observed changes in the immune compartment, we will characterize in deeper the T cell compartment that has been shown to be the main population that predicts a successful response to checkpoint blockade immunotherapy. For that we will apply new algorithm to our already acquired dataset (umap, vortex, etc). We will also confirm the data in a new cohort and we will evaluate new functional markers and repertoire on CD8+ T cells and Tregs. The TCR repertoire profiling was already validated by Hogan et al.
2. We will characterize the innate compartment, more specifically in the unexplored neutrophil compartment. For this point, we already validated our CyTOF panel that allows us to evaluate the different neutrophil compartment. Furthermore, since we observe higher frequency of CD14+CD16-HLA-DRhi monocytes (Krieg et al, 2018) we will also evaluate in deep the monocyte compartment, including activation markers.
3. Since the Fc region of immunoglobulin G (FcγR) could have functional consequences in the binding/digestion of the cells that are bound to human antibodies eg. Ipilimumab and Nivolumab we will evaluate the polymorphism of the FcγRs CD16/CD32 in a new cohort of patients.
4. We will use the knowledge acquired in Aims 1 and 2 to analyze the adaptive immune and the innate compartment in primary/metastatic tumors by multiplexed confocal imaging (collaboration with Dr. Sergio Quezada) from the tumor cell biobank, to define predictors for disease progression and response to immunotherapy.

We anticipate that this project will result in better understanding of cellular and molecular mechanisms within the tumor microenvironment as well as circulating immune cells, which determine the outcome of cancer therapies. Moreover, it may allow us to improve existing therapies by controlled activation or elimination of certain components of the innate compartment.