Tumor size on subsequent days after CT26 inoculation (left), the mean tumor size on day 30 (right). partially negated by depletion of CD8+ cells. Furthermore, the antitumor effects induced by local injection of iMSC/CCL19 were augmented by additional therapy with anti-programmed death (PD)-ligand 1 (PD-L1) antibody, but not with anti-PD-1 antibody. This combination therapy cured most of the tumors in CT26-bearing mice. Conclusion These results suggest that local therapy with iMSC/CCL19 can suppress tumor growth via effective recruitment of CCR7+ DC into tumor sites and increase IFN-+ CD8+ T cells, and that combination with anti-PD-L1 antibody therapy can be a powerful anticancer therapy. strong class=”kwd-title” Keywords: cell engineering, dendritic cells, immunotherapy, programmed cell death 1 receptor, tumor microenvironment Background Mesenchymal stem/stromal cells (MSC) are multipotent cells that can differentiate into osteoblasts, chondrocytes, and adipocytes.1 2 Therefore, these cells show potential as a source for cell therapy. Although the cell surface markers of MSC require further elucidation, highly purified MSC KRN2 bromide can be isolated from adult mouse bone marrow.3 Several studies have reported that MSC accumulate to injured areas and hypoxic tumor microenvironments.4 Taking advantage of these features, MSC have been Adamts5 employed as tumor-accumulating cells for anticancer therapy in various mouse models.5C9 Although several studies have combined human MSC and immunodeficient mice, few studies have developed models with mouse MSC and syngeneic mouse tumors. Syngeneic tumor models are critical for investigating in vivo antitumor T cell immunity after MSC therapy. Chemokine (C-C motif) ligand 19 (CCL19) attracts T cells and dendritic cells (DC) through its receptor C-C chemokine receptor type 7 (CCR7),10 11 thereby regulating cell homing and adaptive immunity.12 13 The expression of CCL19 in human tumors correlates with intratumoral accumulation of CD8+ T cells and patient survival.14 15 In addition, CCL19-producing chimeric antigen receptor (CAR) T cells and endothelial progenitor cells can provide effective anticancer therapies.16 17 In recent years, immune checkpoint blockade (ICB) antibody therapy has received attention as a promising anticancer treatment.18 19 Several ICB antibodies targeting programmed death-1 (PD-1), PD-1 ligand (PD-L1), and cytotoxic T-lymphocyte associated protein 4 (CTLA4) can induce antitumor effects in certain cancer patients.20C22 Given that ICB therapy targeting PD-1 and PD-L1 is likely to restore exhausted antitumor T KRN2 bromide cells in tumor sites, the presence of T cells in tumor tissues is essential for ICB therapy. Indeed, T cell infiltration KRN2 bromide in tumor sites is correlated with the response to anticancer immunotherapy.23 Although promising, the therapeutic efficacy of ICB therapy is limited. Therefore, new strategies are needed to enhance the therapeutic efficacy of ICB. Given that success in anticancer ICB therapy is based on the premise of tumor-infiltrating immune cells, including T cells and DC, MSC-mediated local production of CCL19 could promote the infiltration of those cells and exert an antitumor effect. In this study, we prepared immortalized murine MSC (iMSC) that produce CCL19 (iMSC/CCL19) and investigated their therapeutic efficacy using a CT26 colon carcinoma mouse model. Co-injection of iMSC/CCL19 into mice suppressed the in vivo growth of CT26 compared with that of CCL19-expressing immortalized fibroblasts (iFib/CCL19) in a T cell-dependent manner. In a therapeutic model, local injection of iMSC/CCL19 suppressed CT26 tumor growth; KRN2 bromide furthermore, T cell and DC infiltration increased in mice treated with iMSC/CCL19, but not with iMSC. Moreover, local injection of iMSC/CCL19 augmented the antitumor effects by combination therapy with anti-PD-L1 antibody, but not anti-PD-1 antibody, and this combination therapy cured most CT26-bearing mice. Materials and methods Mice BALB/c 6-week-old female mice were purchased from CLEA Japan (Tokyo, Japan) and maintained under specific pathogen-free conditions. The experiments were carried out according to the Ethical Guidelines for Animal Experiments of the Shimane University Faculty of Medicine (IZ28-72, IZ28-55, IZ29-42, and IZ30-131). Isolation of MSC and Fib Murine MSC were isolated from the bone marrow of BALB/c 6-week-old female mice as described previously.3 Crushed bones from femurs and tibias were treated with 0.2% collagenase (Wako Chemicals USA,) in Dulbecco’s Modified Eagle Medium (DMEM) for 1 hour at 37C. Then, the cell suspension was filtered through a cell strainer (Falcon 2350). The cells were suspended in Hanks balanced salt solution (HBSS) and incubated for KRN2 bromide 30 min on ice with the following monoclonal antibodies (mAbs) (purchased from eBioscience): biotinylated anti-PDGFR, FITC-conjugated anti-Sca-1, PE-conjugated anti-CD45, and PE-conjugated anti-TER119. Biotinylated antibodies were visualized with APC-conjugated streptavidin (Invitrogen). PDGFR+ Sca-1+ CD45?TER119? cells were sorted by a triple-laser Moflo (Dako). Murine primary fibroblasts were isolated from back skin; 10 mm diameter skin.