Supplemental Figure S1. Tumors harvested on treatment d 1 and d 7 were assessed by IHC for P-Neu Y1248. Representative images are shown. Original magnification, 400X. N = 5. Histological analysis of H&E-stained tumor sections. Tumors were collected on treatment day 2 (i.e., 1 day after treatment {plus minus} lapatinib). Representative images are shown. N = 5. Original magnification was 400X. Tumors harvested on treatment d 2 were assessed by IHC for CD3 and FoxP3. Representative images are shown. Original magnification, 400X. N = 5. Tumors harvested on treatment d 2 were assessed by IHC for CD3 and FoxP3. Representative images are shown. Original magnification, 400X. N = 5. RT-qPCR analysis of tumor RNA harvested at treatment d 7 measuring relative levels of indicated gene transcripts. Values were calculated using the ddCT method. Each data point represents the average value of 5 technical replicates, N = 5 tumors. For each transcript, values were corrected for the average value measured in vehicle-treated samples. Student's T-test.
ARTICLE ABSTRACT
Efferocytosis is the process by which apoptotic cells are cleared from tissue by phagocytic cells. The removal of apoptotic cells prevents them from undergoing secondary necrosis and releasing their inflammation-inducing intracellular contents. Efferocytosis also limits tissue damage by increasing immunosuppressive cytokines and leukocytes and maintains tissue homeostasis by promoting tolerance to antigens derived from apoptotic cells. Thus, tumor cell efferocytosis following cytotoxic cancer treatment could impart tolerance to tumor cells evading treatment-induced apoptosis with deleterious consequences in tumor residual disease. We report here that efferocytosis cleared apoptotic tumor cells in residual disease of lapatinib-treated HER2+ mammary tumors in MMTV-Neu mice, increased immunosuppressive cytokines, myeloid-derived suppressor cells (MDSC), and regulatory T cells (Treg). Blockade of efferocytosis induced secondary necrosis of apoptotic cells, but failed to prevent increased tumor MDSCs, Treg, and immunosuppressive cytokines. We found that efferocytosis stimulated expression of IFN-γ, which stimulated the expression of indoleamine-2,3-dioxegenase (IDO) 1, an immune regulator known for driving maternal-fetal antigen tolerance. Combined inhibition of efferocytosis and IDO1 in tumor residual disease decreased apoptotic cell- and necrotic cell-induced immunosuppressive phenotypes, blocked tumor metastasis, and caused tumor regression in 60% of MMTV-Neu mice. This suggests that apoptotic and necrotic tumor cells, via efferocytosis and IDO1, respectively, promote tumor ‘homeostasis’ and progression.
These findings show in a model of HER2+ breast cancer that necrosis secondary to impaired efferocytosis activates IDO1 to drive immunosuppression and tumor progression.
