Authors:
 Taghreed Hirz1,2,3*, Shenglin Mei4*, Youmna Kfoury1,2,3, Chin-Lee Wu5, Shulin Wu5, Douglas M. Dahl5, Philip J. Saylor5, Peter V. Kharchenko2,4, Ninib Baryawno1,2,3,6, David B. Sykes1,2,3
 
 
 Affiliations:
 1Center for Regenerative Medicine, Massachusetts General Hospital, Boston, MA, USA 2Harvard Stem Cell Institute, Cambridge, MA, USA 3Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA 4Department of Biomedical Informatics, Harvard Medical School, Boston, MA, USA 5Massachusetts General Hospital Cancer Center, Harvard Medical School, Boston, USA 6Childhood Cancer Research unit, Dep. Of Children’s and Women’s Health, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden
 
 
 Abstract Body:
 "Advances in immune checkpoint blockade therapy have improved the treatment paradigm for many cancer types, yet unselected prostate cancer generally exhibits de novo resistance to immunotherapy. This inherent resistance appears to be due, at least part, to the immune-suppressive nature of the tumor microenvironment (TME). It remains unclear how to therapeutically overcome this hurdle and render tumors sensitive to immunotherapy.
 
 Fresh prostate tissues were collected from prostatectomy cases of 20 treatment-naïve patients, obtaining both tumor and adjacent benign tissue. We have examined the cellular composition of the prostate TME at the single-cell level across a spectrum of primary tumor grades and stages (Gleason 6-10; pathologic T-stage 2a to 3b) using droplet-based single-cell RNA sequencing. 
 
 Analysis of transcriptional states revealed a complex TME with the major cell populations including immune cells and non-immune stroma. Unsupervised clustering for myeloid cells revealed 3 subpopulations of monocytes, 3 of macrophages and monocytic dendritic cells. Tumor samples showed enrichment in M2-tumor-associated macrophages (TAM), a finding confirmed with multiplex-immunohistochemistry, and particularly in cases of high Gleason scores. Monocyte subpopulation 2 (Monocytes-2) showed high expression for Myeloid Derived Suppressor Cell (MDSC)-centric genes, suggesting an immune suppressive function.
 
 Detailed analysis of the lymphocyte compartment revealed the expected subsets, CD4+ T-helper and T-regulatory (Treg) cells, CD8+ cytotoxic T-lymphocytes (CTLs), as well as NK and NKT cells. Two subpopulations of CD8+ CTLs were obtained (CTL-1, CTL-2). The relative abundance of CD8+ CTLs, particularly CTL-2, gradually decrease from normal samples towards tumor samples with high Gleason scores. 
 
 
 Interestingly, tumor samples exhibited strong correlation between the functional states of these T-cell populations, in particular CTL-2 exhaustion and Treg activity, with MDSC immune suppressive gene signature of Monocyte-2. Screening the possible ligand-receptor interactions between Monocyte-2:CTL-2 and Monocyte-2:Tregs identified 12 and 16 potential channels of interaction, respectively.
 
 Based on this, we aim to target these suppressive myeloid population using targeted differentiation therapy, to push the cells towards mature effector myeloid cells. In vivo data using the DHODH inhibitor brequinar (BRQ), a drug that overcomes myeloid differentiation arrest in leukemia models, significantly (1) improved the survival probability of prostate tumor bearing mice, (2) reduced the prostate tumor growth, (3) reduced MDSC cell proportion, and (4) increased effector macrophages. 
 
 In conclusion, our data presents single-cell analysis of prostate tumor immune environment. Computational analyses point to correlation between T-cell exhaustion and myeloid immune suppressive function; this correlation represents a potential therapeutic target to sensitize tumors to immunotherapy and to reduce future patient suffering due to advanced prostate cancer."
 
 
 Funding Acknowledgments:
 We are particularly indebted to our patients and their clinical care teams. We acknowledge funding from Dana-Farber / Harvard Cancer Center Nodal Award (CCSG grant P30CA006516). We gratefully acknowledge support from Bill & Cheryl Swanson and from Gunther & Maggie Buerman.
 
 
 Corresponding Author Email:
 thirz@mgh.harvard.edu
 
 
 Conflict of Interest:
 P.V.K. serves on the Scientific Advisory Board to Celsius Therapeutics Inc. D.B.S. is a co-founder and holds equity in Clear Creek Bio, holds equity in SAFI Biosolutions and is a consultant to Keros Therapeutics.

Single-cell genomic profiling reveals a myeloid immunosuppressive microenvironment in human prostate cancer