Abstract A135: Combining low-dose chemotherapy with an NK cell-based immunotherapy as a treatment for triple-negative breast cancer
Authors: Idri Sarra , Pawelec Graham , Barnett Yvonne , Pockley Graham 
Published: February 2019

Abstract
In addition to being less toxic and having a lower impact on a patient’s quality of life, evidence suggests that low-dose or metronomic chemotherapy modulates adaptive and innate antitumor immune responses. In this study, we examined whether the treatment of breast cancer cells and animals bearing breast cancer cell-derived tumors with low, nontoxic doses of chemotherapies promotes sensitivity to natural killer (NK) cells, based on target cell killing in vitro and the control of tumor growth and metastasis in vivo. We have developed a low-dose doxorubicin treatment protocol that sensitizes triple-negative breast cancer (TNBC) cells (MDA-MB-231, MDA-MB-468) to killing by NK cells. Specifically, low-dose doxorubicin treatment induced a “senescent-like” state in breast cancer cells concomitant with an impaired proliferative capacity, as shown by a decreased expression of Ki-67. Treatment of breast cancer cells also upregulated their expression of ligands for activatory NK cell receptors (e.g., MICA/B, ULBP1, ULBP2, ULBP3) and markedly increased their sensitivity to lysis by donor-derived, primary NK cells and the NK-92 cell line, as assessed using an in vitro flow cytometry-based assay. The cytotoxicity of donor-derived NK cells was markedly increased by prior stimulation with IL-2. The therapeutic potential of low-dose chemotherapy alone and in combination with adoptively transferred resting and activated NK cells was evaluated using immunodeficient NSG mice bearing MDA-MB-231/RFP/LUC-derived human TNBC xenografts. Although low-dose doxorubicin treatment alone reduced the growth of the primary tumors, as assessed using caliper measurements and in vivo imaging, the growth of the primary tumor was more markedly reduced following the adoptive transfer of healthy-donor derived NK cells (mean tumor volume: 70.56mm3 versus 228.8mm3 in control groups at day 49). Importantly, although treatment with low-dose doxorubicin alone also delayed the onset of metastasis by 7 days (day 49 in control animals, day 56 in treated animals), no signs of metastasis were observed in healthy-donor derived NK cell-treated animals at the time of culling on day 50. The expression of MICA/B was 8.5 times greater on tumor cells derived from mice that received the combination treatment (p=0.0001) and these cells were more sensitive to killing by NK-92 cells in vitro (3-fold increase at E:T 1:5). Furthermore, in vitro studies demonstrating that the treatment of MDA-MB-231 cells with low-dose doxorubicin reduces the number of CD44High/CD24−/low/EpCAM+ Cancer Initiating Cells (CICs) were confirmed by there being a significantly lower number of CICs in tumors derived from treated mice. These novel findings indicate that this approach has the capacity of preventing the primary tumor “fueling” the dissemination of aggressive, metastatic disease. Taken together, these studies demonstrate that the treatment of breast cancer with low, nontoxic doses of chemotherapies promotes their sensitivity to killing by NK cells in vitro and in vivo. We have shown that administering human NK cells to mice bearing human breast tumors that have been treated with low-dose doxorubicin reduces the growth and metastasis of tumors and eliminates the aggressive cells that “feed” this process. Ongoing studies are further interrogating the influence of this approach on breast cancer metastasis. In summary, this strategy offers a promising opportunity for more effectively treating patients with aggressive, triple-negative breast cancer.
Citation Format: Sarra Idri, Graham Pawelec, Yvonne Barnett, Graham Pockley. Combining low-dose chemotherapy with an NK cell-based immunotherapy as a treatment for triple-negative breast cancer [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A135.