Abstract B54: Single-cell analytics in claudin-low TNBC reveals immunostimulatory effects of low-dose metronomic cyclophosphamide treatment
Authors: Singh Swarnima , Wan Ying-Wooi , Lee Nigel , Kim Se-Jin , Liu Zhandong , Perou Charles M. , Zhang Xiang , Rosen Jeffrey 
Published: March 2020

Breast cancer is a heterogeneous disease. It can be classified into at least five subtypes: luminal A and B, normal breast like, Her2 positive, basal like, and claudin low. Among these, claudin-low tumors have relatively poor prognosis. These tumors are characterized by the low expression of cell-cell junction proteins along with an enrichment of EMT markers. They are more aggressive and the rate of response to primary chemotherapy is around 32%. Initial data have shown that murine p53 null claudin-low tumors are only sensitive to cyclophosphamide treatment. Cyclophosphamide (CTX) is a chemotherapy drug that has been used in lymphoma, leukemia, and some solid tumors. At low and continuous doses, it shows immunostimulatory properties. In this study we describe the implementation of a novel combination treatment for claudin-low TNBC as informed by combined scRNA-seq and V(D)J seq. Immune profiles of a murine claudin-low tumor model (T12) shows heavy infiltration by tumor-associated macrophages (TAMs) pretreatment. Initial data in T12 tumors also suggest that CTX induces CD4+ and CD8+ T-cell homing to the primary tumor. Wild-type Balb/c mice implanted with T12 tumors respond favorably to CTX treatment as compared to T cell-deficient NSG mice. We combined 5´ scRNA-seq with V(D)J seq to gain an insight into how immune cell subpopulations change during the course of treatment. Combining tSNE plots for untreated and treated tumors revealed the emergence of new transcriptionally distinct populations in the CTX treated group, which included six T cell subpopulations. Projection of T cells along pseudotime using Monocle3 revealed a shift from naive CD8+ and CD4+ T cells to a terminal effector CD8+ T-cell phenotype and CD4+ effector memory cells in treated tumors while untreated tumors have higher numbers of exhausted Tigit+ CD8+ T cells and Tregs. V(D)J analysis revealed higher numbers of unique TCRs in CTX-treated tumors and greater clonal expansion of T cells. However, we noted a persistence of TAMs in our model even after treatment as well as high recurrence rates upon cessation of treatment. We then examined markers specific to the TAM subsets in the treated and untreated tumors and observed that TAMs expressed high levels of Csf1r. A chow containing PLX3397(iCSF1R) was employed to inhibit CSF1R, a macrophage differentiation/recruitment factor. Combination treatment with CTX and the iCSF1R chow lead to long-term regression of T12 tumors. Rechallenging these mice resulted in much slower tumor growth, indicating the formation of T cell memory. scRNA-seq and V(D)J seq is being performed on these samples to identify the clonal dynamics of effector T cells that assist in this striking regression. We hypothesize that CTX treatment can sensitize T12 primary tumors to T cell-mediated apoptosis, and that TAMS can suppress T-cell function and promote chemoresistance. Furthermore, depleting these TAMs in combination with CTX treatment may lead to long-term durable regression of both the primary tumor and metastases.

Citation Format: Swarnima Singh, Ying-Wooi Wan, Nigel Lee, Se-Jin Kim, Zhandong Liu, Charles M. Perou, Xiang Zhang, Jeffrey Rosen. Single-cell analytics in claudin-low TNBC reveals immunostimulatory effects of low-dose metronomic cyclophosphamide treatment [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B54.