Abstract PR07: Characterization of human effectors in the tumor microenvironment of NSCLC patients and lung tumor xenograft models
Authors: Ra Hyun-Jeong , Brake-Silla Gisela , Eruslanov Evgeniy , Sharp Michael , Deshpande Charuhas , Feldman Michael D. , Ziober Amy , Wang Li-Ping , Sepulveda Manuel A. , Snyder Linda A. , Danet-Desnoyers Gwenn 
Published: May 2016

Abstract
Non-small cell lung cancer (NSCLC) is one the most prevalent forms of lung cancer. Lung tumors consist of a heterogeneous population of tumor cells, immune cells and stromal cells. It is critical to assess the nature and localization of immune effector cells and their expression of immune checkpoint molecules to develop more effective therapeutic strategies. In this study, we used immunohistochemistry (IHC) to assess the localization of human immune cells and expression of immune checkpoint molecules to define the microenvironment composition. Furthermore, we developed a mouse xenograft model of human lung tumor using BLT-NSG mice with a reconstituted human immune system and A549 cells.
Formalin-fixed paraffin embedded NSCLC lung tumor sections (n=28 adenocarcinoma, n=16 squamous) were processed by IHC to assess expression and localization of CD4, CD8, MPO, Foxp3, CD68 and cytokeratin, as well as immune checkpoint molecules (PD-1, Tim-3, LAG-3) and ligands (PD-L1 and VISTA). For the xenograft model, BLT-NSG mice (NSG mice transplanted with fetal liver CD34+ cells and autologous thymus) at 12-16 weeks post-transplant were injected with A549-luciferase (A549-Luc) cells via transpleural or intravenous injection. Weekly bioluminescence imaging was done to assess tumor growth. Lungs and spleens were harvested and enzymatically dissociated for flow cytometric analysis of human effectors and immune checkpoint molecules.
We found that significantly more human effectors are localized to the stromal compartment (cytokeratin-) than within the cytokeratin+ tumor nests. The stromal compartment contained 12-fold more CD8+ T-cells, 23-fold more CD4+ T-cells, and 11-fold more regulatory T-cells (Foxp3+). Two-fold more neutrophils (MPO+) and macrophages (CD68+) were found in stroma outside of tumor nests. There were significantly more CD8+ T-cells (p&lt;0.05) and MPO+ neutrophils (p&lt;0.01) localized within the cytokeratin+ areas of squamous compared to adeno tumors. PD-1, Tim-3 or LAG-3 IHC showed that 100-fold more PD-1+ cells are found within the tertiary lymphoid structures in the stromal compartment. 19-fold more Tim-3+ cells, both lymphocytes and macrophages, are found in the stromal compartment. LAG-3+ cells are lymphocytes, and localized mostly to the stromal compartment (11-fold more than in cytokeratin+ areas). PD-L1 IHC showed that 12/16 (75%) of squamous tumors and 14/28 (50%) of adeno tumors express PD-L1 on tumor cells, and H-Scores of the PD-L1 staining from squamous tumors were significantly higher than the adeno tumors (p=0.019). A majority of macrophages expressed PD-L1, but the expression level varied from sample to sample (1+ to 3+ scoring). VISTA IHC showed that lymphocytes, neutrophils and macrophages expressed VISTA, and 26-fold more VISTA+ cells are found in the stromal compartment.
In our A549 xenograft model study, NSG and BLT-NSG mice with IV-injected A549 cells showed tumor growth differences by bioluminescence imaging. Flow cytometric analyses showed an increased percentage of non-naïve CD8+ (88%) and non-naïve CD4+ (97%) cells compared to spleens (44% non-naïve CD8+ cells and 74% non-naïve CD4+ cells). There was an increased expression of LAG-3 in both human CD8+ (19-90%) and human CD4+ (9-61%) non-naïve T-cells in the lungs, but LAG-3+ cells in the spleen were minimal (&lt;6%). PD-1 expression was high in non-naïve T-cell population in both lungs and spleen (&gt;70%). In addition, there was an up-regulation of PD-L1+(4%) and VISTA+ (12%) in the human CD14+ population in the lungs. In conclusion, the expression of various immune checkpoint molecules in effector cells may limit their localization within the stromal compartment. Furthermore, our xenograft model was able to recapitulate our observations with human patient samples, and would be a valuable in vivo mouse tool to test new therapeutic agents.
Citation Format: Hyun-Jeong Ra, Gisela Brake-Silla, Evgeniy Eruslanov, Michael Sharp, Charuhas Deshpande, Michael D. Feldman, Amy Ziober, Li-Ping Wang, Manuel A. Sepulveda, Linda A. Snyder, Gwenn Danet-Desnoyers. Characterization of human effectors in the tumor microenvironment of NSCLC patients and lung tumor xenograft models. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr PR07.