Abstract 4064: Spatial re-distribution of natural killer cells targets drug resistance and anti-tumor response, <i>ex-vivo</i>
Authors: Munisha Smalley, Basava U. Shanthappa, Hans Gertje, Mark Lawson, Manjusha Biswas, Saravanan Thiyagarajan, Biswanath Majumder, D. C. Doval, Anurag Mehta, Neyaz Alam, Nabendu Murmu, S. P. Somashekhar, Aaron Goldman
Journal: Cancer Research (2018)

Abstract Background: It is now clear that response or resistance to therapy is contingent on the tumor microenvironment, which is comprised of malignant cells, normal stroma, and immune landscape; attributes that are unique to each individual patient. This is particularly true for emerging anticancer drugs, such as immune checkpoint inhibitors, which recalibrate the body's own immune defense largely by modulating exhaustion of cytotoxic lymphocytes including T cells and natural killer (NK) cells. However, clinical response to therapy varies enormously. There is a critical gap in our understanding for the mechanisms that drive response or resistance to conventional drugs and immunotherapies at the individual patient level. Methods: Here, we used in-vitro and in-vivo experiments to study the role of NK cells in models of drug tolerance. Next, we employed (CANscriptTM), a clinically-validated ex-vivo tumor model that recreates and preserves the native, patient tumor microenvironment, which integrates an algorithm-driven method to predict clinical response to therapy (M-Score). Utilizing tissue from patients diagnosed with luminal, HER2 positive, and triple-negative (ER- PR- HER2-) breast cancers (N=10), we studied spatial heterogeneity of the tumor-immune contexture, M-Score, and phenotypic alterations under pressure of conventional standard-of-care regimens and immunotherapies including immune-checkpoint inhibitors. To do this, we used a comprehensive panel of immunological assays to evaluate changes in cytotoxic lymphocytes by flow cytometry, immunohistochemistry and cytokine profiling (i.e. CD56, MHC class 1A/B, NKG2D/C, CD8, CD3, PD-1, CTLA-4, TIM-3, LAG-3, 4-1BB, granzyme A/B). Results: We identified that drug resistance is associated to downregulation of activating proteins on NK cells including NKG2D/C, reducing their cytotoxic capability. Furthermore, tumor response, predicted by M-Score, correlates to a unique pattern of spatially-distributed NK cells, which associated a pro-inflammatory cytokine signature from the tumor microenvironment. Interestingly, we determined that therapy-induced expression of exhaustion biomarkers on NK cell exhaustion inversely correlated to the expression of cytotoxic granzyme B in the tumor microenvironment. Conclusions: Taken together, these data demonstrate an integral role that NK cells contribute to resistance and the antitumor response to therapy including both conventional and immuno-modulatory drugs. We further demonstrate that CANscriptTM can be harnessed to study response and resistance of the innate and adaptive immune system. Such an advance in our preclinical methods to study anticancer drugs at the individual patient level can help guide treatment decisions for clinicians while simultaneously functioning as a platform to study clinical efficacy of novel and emerging agents. Citation Format: Munisha Smalley, Basava U. Shanthappa, Hans Gertje, Mark Lawson, Manjusha Biswas, Saravanan Thiyagarajan, Biswanath Majumder, D. C. Doval, Anurag Mehta, Neyaz Alam, Nabendu Murmu, S. P. Somashekhar, Aaron Goldman. Spatial re-distribution of natural killer cells targets drug resistance and anti-tumor response, ex-vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4064.