Abstract 2451: Dormant mammary tumors resist antigen-specific killing and regulate systemic immunity in association with PTN and DKK3
Authors: Trotter Timothy N. , Serra Delila , Dagotto Carina , Wang Tao , Yang Xiao , Wei Junping , Lei Gangjun , Lyerly H. Kim , Hartman Zachary C. 
Published: June 2022

Abstract
Little is known regarding why the immune system is ineffective at eliminating certain disseminated/dormant tumor cells before overt metastatic recurrence. Previous findings revealed that the adaptive immune system reduces growth of proliferative D2A1 tumors in Balb/c versus SCID mice while dormant D2.1 tumors are largely unaffected by adaptive immunity, despite being significantly infiltrated by T cells. Therefore, the aim of the present study was to determine the downstream effects and molecular underpinnings of dormancy-mediated alterations to the immune environment. Experiments were performed using two independent clones of a spontaneous mammary tumor from a female Balb/c mouse that display varying degrees of dormancy in vivo. D2A1 cells grow rapidly upon orthotopic or systemic injection while D2.1 cells remain dormant long term in local and metastatic sites. Both cell lines were modified to express enhanced green fluorescent protein (eGFP) for use with Just eGFP Death Inducing (JeDI) T cells which contain a T cell receptor (TCR) targeted to eGFP200-208. Adoptive transfer of JeDI T cells caused complete rejection of D2A1-eGFP tumors in the mammary fat pad (MFP), while no difference in growth was observed in D2.1-eGFP tumors. Interestingly, contralateral MFP implantation of D2.1-eGFP tumors resulted in a partial rescue of D2A1-eGFP rejection suggesting systemic immune suppression by distant, dormant tumor cells. Bulk RNA-sequencing of D2A1 and D2.1 tumors revealed significant upregulation of pathways associated with immune activation and extracellular matrix (ECM) remodeling in dormant tumors. Further, Gene Set Enrichment Analysis (GSEA) showed an enrichment for signatures of mammary stem cells and luminal progenitor cells, as well as exhausted vs memory CD8 cells and T cell anergy, in D2.1 tumors. Of the top upregulated genes in D2.1 tumors, pleiotrophin (PTN) and Dickkopf WNT Signaling Pathway Inhibitor 3 (DKK3) were chosen for further investigation. Overexpressing PTN or DKK3 in D2A1 cells significantly increased MFP tumor growth in Balb/c but not SCID mice. In addition, PTN or DKK3 overexpression altered both local and systemic T cell profiles, including decreased effector memory (CD62l-, CD44+) and increased naïve (CD62l+, CD44-) CD4 and CD8 cells. Humoral immunity was also affected, as indicated by decreased eGFP-specific antibodies in serum from mice bearing PTN or DKK3 overexpressing tumors. Conversely, knocking down PTN or DKK3 in D2.1 cells had the opposite effect on systemic immunity. Taken together, these data indicate that dormant tumors can resist antigen-specific killing by CD8 T cells despite high T cell infiltration, and possibly suppress antigen-specific immunity in distant tumor sites. Moreover, both PTN and DKK3 emerge as potential mediators of dormancy-induced immune evasion.
Citation Format: Timothy N. Trotter, Delila Serra, Carina Dagotto, Tao Wang, Xiao Yang, Junping Wei, Gangjun Lei, H. Kim Lyerly, Zachary C. Hartman. Dormant mammary tumors resist antigen-specific killing and regulate systemic immunity in association with PTN and DKK3 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2451.