In situ tumor arrays reveal early environmental control of cancer immunity [NGS3077]

The immune phenotype of a tumor is a key predictor of its response to immunotherapy. Patients who respond to immune checkpoint blockade generally present with tumors infiltrated by T cells, a phenotype referred to as ‘inflamed’. However, not all inflamed tumors respond to therapy, and even lower response rates occur among patients with tumors that lack T cells (‘desert’) or that spatially exclude T cells to the periphery of the tumor lesion (‘excluded’). Despite the importance of these tumor immune phenotypes in patients, little is known about their development, heterogeneity or dynamics due to the technical difficulty of modeling and tracking these features in situ. Here, we introduce STAMP (skin tumor array by microporation), a preclinical approach that combines in vivo high-throughput time-lapse imaging with next generation sequencing of tumor arrays.  Using this approach, we follow the early formation of thousands of tumors to show that development of a given immune phenotype is not under strict control of tumor genetics or transcriptional state, but is also influenced by local features of the tumor microenvironment. Only the spatial organization of T cells, specifically early infiltration of T cells recruited by fibroblasts and monocytes into the core of a tumor, was predictive of functional T cell attack and tumor rejection. Evaluating the dynamic immune-history of tumors revealed that early conversion to the immune inflamed phenotype was predictive of therapy-induced or spontaneous tumor regression. Thus, STAMP captures the dynamic and complex relationships of spatial, cellular and molecular components of tumor progression and has the potential to translate therapeutic concepts into successful clinical strategies. Deciphering the molecular regulators of tumor T cell infiltration in the pre-clinical STAMP (Skin Tumor Array by Microporation) model. The classification of immune infiltrated, immune excluded or immune desert tumors is a powerful predictor of clinical response to immunotherapy. Patients who respond to immune checkpoint blockade generally present with tumors that are infiltrated by activated T cells. However, the majority of patients present with tumors that are poorly (immune desert) or improperly (immune excluded) infiltrated by CD8 T cells. In order to mimic the clinical observations and perform live Imaging of the Immune cell infiltration in tumors, we developed a new pre-clinical mouse model called STAMP (Skin Tumor Array by Microporation). We use a laser-assisted technique to deposit tumor cells in a regular pattern into the superficial dermis of the ear of the animal. When GFP expressing KPP tumor-cells are implanted into a CD4-Cre Tdtomato mouse, we can perform live imaging of TdTomato+ T Cells trafficking in and out of each micro-tumor of the array. Using live imaging we were able to classify each of the implanted tumors as: 1 Hot/Inflamed tumors (GFP+ tumors infiltrated with T cells in the tumor core), 2-Excluded Tumors (GFP+ tumors with T cells segregated around the tumor area) 3-Resolved tumors (no GFP+ tumor cells anymore but only a cloud of T cells) or 4-Desert tumors (GFP+ tumor cells without any T cells infiltration). Even though the KPP mother tumor cell line used for the implantation is derived from a single cell clone, the skin microenvironment is able to dictate the infiltration pattern of individual tumors implanted into the same recipient. Even 2mm apart, those tumors behave as independent compartments. As the tumors are implanted in the superficial dermis, we were able to biopsy them out and separate them by categories (Resolved, Inflamed, Excluded and Desert). We harvested RNA directly from biopsies of individual tumors of each category. We performed this experiment 7 times independently, and have a total of 33 samples (cf below). We wish to characterize the differences in Transcriptome profile to determine regulators, drivers and potential targets of these 4 tumor categories. KPP GFP+ tumor cells were implanted into the ear of a B6.Cg-Foxn1nu/J (JAX: 000819) animal with the STAMP technic. The same day naïve TdTomato T cells (colony 7976; CD4.cre.tg_Rosa26.LSL.tdTomato.cki_OT-I.TCR.tg) were adoptively transferred i.v into the same mouse. Tumor infiltration is imaged from day 5-14 of each individual tumor. Day 13 after implantation, we performed live imaging of the whole Tumor array to be able to annotate tumors as Rejected, Inflamed, Excluded and Desert. 1mm Skin tumor biopsies were harvested and pooled by category (Resolved, Hot/Inflamed, Excluded and Desert) or treated individually.