Abstract PR11: Dissecting mechanisms of PD-1 blockade with single-cell RNA-sequencing
Authors: Miller Brian C. , Wadsworth Marc H. , Bi Kevin , Hughes Travis K. , Sharpe Arlene H. , Shalek Alex K. , Haining W. Nicholas 
Published: March 2017

Abstract
Anti-PD-1 therapy is an important new treatment option for many different types of malignancies, but overall response rates are less than 40%. We do not yet understand which patients will benefit and what resistance mechanisms allow tumor escape. The goal of this work is to understand the mechanisms by which anti-PD-1 therapy augments the anti-tumor immune response at the cellular level. Given that anti-PD-1 therapy is thought to work by altering the immunosuppressive tumor microenvironment, efforts to improve its efficacy will require a deep understanding of this complicated milieu. This will require analysis of thousands of cells using methodology that avoids the pitfalls of current techniques that have either limited scope - flow cytometry, immunohistochemistry - or limited resolution - bulk RNA sequencing. To this end, we have developed a massively parallel single-cell RNA-sequencing platform (Seq-Well) that comprehensively defines the global expression profile of all major immune lineages in the tumor microenvironment. Seq-Well uses a fabricated chip with nearly 100,000 nanowells into which barcoded beads and individual cells are distributed prior to lysis and RNA capture. Mice were implanted with two different transplantable models of cancer (MC38 colon carcinoma or B16 melanoma) and treated with anti-PD-1 or control antibodies. Tumors were harvested and CD45+ tumor-infiltrating leukocytes isolated by FACS. Thousands of cells were sequenced using Seq-Well with a median recovery of approximately 1,000 genes/cell. This level of expression diversity allows us to clearly distinguish different cell populations within the tumor microenvironment. We detect two transcriptionally distinct populations of CD8+ T cells, one that is highly proliferative (as marked by Ki-67), and one that has higher expression of perforin and TIM-3. The Ki-67+ population is enriched for a gene expression signature characterized by effector CD8+ T cells early in viral infection, consistent with their more proliferative nature. We hypothesize that this cluster of CD8+ T cells is also more functional given this signature enrichment and its lower expression of TIM-3, a marker found on exhausted CD8+ T cells. Comparisons of anti-PD-1 treated and control treated tumors are ongoing. In conclusion, massively parallel single-cell RNA-sequencing is a promising technology for the analysis of tumor immune infiltrates that will allow us to address the mechanisms by which checkpoint blockade controls tumor growth. By advancing our knowledge of an important immune checkpoint therapy, we aim to better understand who will respond to therapy, what resistance mechanisms may develop, and how to augment therapeutic efficacy with additional treatments.
This abstract is also being presented as Poster A79.
Citation Format: Brian C. Miller, Marc H. Wadsworth 2nd, Kevin Bi, Travis K. Hughes, Arlene H. Sharpe, Alex K. Shalek, W. Nicholas Haining. Dissecting mechanisms of PD-1 blockade with single-cell RNA-sequencing. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr PR11.