Evaluating the role of -H4 as a suppressor of tumor infiltrating lymphocytes and a target for immunotherapy in breast cancer Elizabeth C. Wescott1,3, Paula I. Gonzalez-Ericcson, M.D.3, Violeta Sanchez3, Justin M. Balko, Pharm D, Ph.D.1,2,3 Departments of Pathology, Microbiology, and Immunology1, Medicine2, Vanderbilt University Medical Center, Nashville, TN; Breast Cancer Research Program3, Vanderbilt-Ingram Cancer Center; Vanderbilt University [email protected]

Abstract B7-H4 is expressed on EpCAM+ murine mammary cancer cells

• Immunotherapy has seen broad success across cancer types and has become a key aspect of TNBC treatment, but some patients fail to respond A B7-H4+ Murine Cell Lines B • B7-H4 (VTCN1) is an alternative ligand in the CD28/B7 family of 2500 molecules, like PD-L1 2000 • We observed both high and low B7-H4 expression on several mammary cancer cell 1500 1000

lines B7-H4 MFI • B7-H4 is exclusively expressed on cells bearing epithelial markers 500 • mRNA expression data from a variety of human breast cancer cell lines corroborate this 0

4T1 strong positive correlation of B7-H4 expression on epithelial cells E0771 EMT6 PyMT-B6 • B7-H4 may act as an alternative mechanism of immunologic escape in breast cancer. MMTV-NeuMMTV-NIC (A) We screened a panel of murine mammary cancer cell lines and found both high and low Background expression of B7-H4. (B) Those cells that were B7-H4+ were also EpCAM+ in multiple cell lines.

Vtcn1 expression A A B C 4

3

2

1

0 Relative Normalized Expression Normalized Relative

EMT6

MMTV-Neu

MMTV-Neu Epithelial

MMTV-Neu Mesenchymal B B7H4 high B7H4 low (A) The MMTV-Neu cell line consists of epithelial and mesenchymal cell phenotypes. (B) When these cell populations were separated by differential trypsinization, we found only the epithelial E cells expressed B7-H4. (C) These data were further confirmed by quantitative PCR of the heterogenous, epithelial, and mesenchymal cells, compared to an EMT6 negative control.

* C ** B7-H4 expression is not regulated by interferon 100 * **** MMTV-Neu 80 100 B7-H4 60 80 PD-L1

60 40 40 20 % Positive Cells Positive % 20 B7H4 tumor (% B7H4 tumorof (% tumor) 0 0 FI SR MR ID (A) We quantified CD8 T cell infiltration as described by Unstim IFNα STING-NP IFNγ Stimulation Condition (72hr) Gruosso et. al J Clin Invest. 2019. (B) We found both D 100 B7H4 Hi low and high B7-H4 expression in TNBC tumors. (C) We found B7-H4 expression was not induced by Type I or Type II interferon treatment, B7H4 Lo B7-H4 expression was more often on the MR and ID unlike PD-L1. 75 tumors. (D) Tumors with high B7-H4 expression had worse overall survival. (E) We performed digital spatial 50 p = 0.0110 profiling to identify expression in different Conclusions & Future Directions regions of interest (ROI) and found PD-L1 and STING 25 expression on tumor cells in a subset of SR tumors. We • Immunotherapy has seen broad success in a variety of tumor types, including also found EpCAM expression on MR tumors, and breast cancer. Percent overallPercent survival 0 CD44 expression on SR tumors. Data generated by • B7-H4 is expressed in breast cancers and correlates with epithelial markers. 0 24 48 72 96 120 Paula Gonzalez-Ericcson, M.D. • B7-H4 expression is not regulated by interferon stimulation, unlike PD-L1, but Months elapsed may be regulated by mesenchymal to epithelial transition in tumors. VTCN1 expression correlates with epithelial cell markers in human • I will investigate models of enforcing cell transition into EMT and measure breast cancer cell lines changes in B7-H4 expression.

VTCN1 and EpCAM mRNA Expression (Ranks) VTCN1 and CDH1 mRNA Expression (Ranks) CCLE Breast 60 60 A B 1.0 r = 0.475 r = 0.473 2 40 R2 = 0.226 40 R = 0.223 p = 0.0002 p = 0.0002 mRNA (Rank)

mRNA (Rank) 0.5

20 20 B7-H4 CDH1 EPCAM 0 0 0.0 0 20 40 60 0 20 40 60 expression ) VTCN1 mRNA (Rank) VTCN1 mRNA (Rank) VTCN1 and CD44 mRNA Expression (Ranks) VTCN1 and TWIST1 mRNA Expression (Ranks) -0.5 VTCN1 ( 60 60 Correlation to -1.0 r = -0.2987 r = -0.521 40 2 40 2 R = 0.0892 R = 0.271 P N U D p = 0.0253 p = <0.0001 _ _ T T mRNA (Rank)

mRNA (Rank) M M E E 20 20 _ _ B7-H4-H4 N N R

CD44 R

TWIST1 E E L L 0 0 L L O O 0 20 40 60 0 20 40 60 H H • To determine whether B7-H4 is an alternative mechanism of immunologic VTCN1 mRNA (Rank) VTCN1 mRNA (Rank) escape in breast cancer, I will test whether enforced expression of B7-H4 (A) We looked at the CCLE breast dataset and found B7-H4 (VTCN1) expression correlates with epithelial cell markers but not mesenchymal cell markers. (B) expression data from causes EMT6 tumors to be resistant to anti-PD-L1 immunotherapy in vivo. upregulated during epithelial-to-mesenchymal transition (EMT) are negatively correlated with VTCN1 expression.