2 Integrin β1^high/β3^high cancer stem cell-directed targeting of melanoma

<h3>Background</h3> We reported integrin β4 (ITGB4)-targeted immunologic strategies, e.g., ITGB4 protein-pulsed dendritic cell (ITGB4-DC) vaccine and adoptive transfer of anti-CD3/anti-ITGB4 bispecific antibody-armed tumor-draining lymph node T cells. Both immunotherapies demonstrated significant efficacy in inhibiting local tumor growth and lung metastases in murine breast (4T1) and head and neck squamous cell carcinoma (SCC7) tumor models. These therapeutic efficacies were associated with T cell as well as humoral anti-integrin β4^highcancer stem cells (CSCs). In this study, we report targeting integrin β1^high/β3^high CSCs while simultaneously blocking PD-L1. <h3>Methods</h3> The expression of integrin subunits was assessed in murine melanoma cell lines F10 and D5 and in fresh mouse melanoma tumors. We generated integrin subunits β1 (ITGB1) and/or β3 (ITGB3)-pulsed DCs for vaccination and evaluated their antitumor efficacy either alone or in combination with anti-PD-L1 in targeting ALDH^high CSCs <i>vs.</i> ALDH^low non-CSCs. <h3>Results</h3> Integrin subunits β1 and β3 are both expressed in cultured F10 and D5 melanoma cells (figure 1), and in newly harvested fresh melanoma tumors (figure 2). The expression of ITGB1 and ITGB3 on F10 and D5 ALDH^high CSCs (83.3% and 87.1% respectively) are higher than their expression on ALDH^low F10 non-CSCs (0.2%) and ALDH^low D5 non-CSCs (2.0%) (figures 3-5), suggesting that ITGB1 and ITGB3-targeted vaccine is directed towards the ITGB1^high/ITGB3^high melanoma CSCs. ITGB1 and/or ITGB3-pulsed DC vaccine in F10 model demonstrated that ITGB1-DC or ITGB3-DC vaccine each exhibited significant antitumor efficacy, and ITGB1-DC + ITGB3-DC dual vaccine showed an additive/synergistic anti-tumor effect. ITGB1 and ITGB3 loaded on separate DCs demonstrated more potent antitumor efficacy than those loaded on the same DC (figure 6). Anti-PD-L1 augmented the antitumor efficacy of ITGB1-DC/ITGB3-DC vaccine (figure 7). Recent studies suggested that bone marrow (BM)-DCs expressing dysfunctional integrin β2 (ITGB2) exhibited more potent antigen presentation function than WT BM-DCs. We found that ITGB2 highly expressed on the surface of BM-DC (63.3%), but not on F10 or D5 tumor cells. On-going experiments to verify that ITGB1 and ITGB3-targeted vaccines have reduced the number and function of ITGB1^high/ITGB3^high melanoma CSCs <i>via</i> both cellular and humoral mechanisms, and the generation and use of ITGB2 knock-down DCs for the vaccine preparation to improve the efficacy of DC vaccine targeting ITGB1^high/ITGB3^high melanoma CSCs warrant further investigation. <h3>Conclusions</h3> ITGB1-DC or ITGB3-DC vaccine alone each exhibited significant antitumor efficacy, and ITGB1-DC + ITGB3-DC showed additive/synergistic anti-tumor effect. ITGB1 and ITGB3 loaded on separate DCs demonstrated more potent antitumor efficacy than those loaded on the same DCs for vaccination, and anti-PD-L1 significantly augmented such antitumor efficacy.