The immunological effects of chemotherapy-induced tumor cell death are not completely understood. Accumulating evidence suggests that phagocytic clearance of apoptotic tumor cells, also known as efferocytosis, is an immunologically silent process, thus maintaining an immunosuppressive tumor microenvironment (TME). Here we report that, in the breast tumor microenvironment, thymosin α-1 (Tα-1) significantly reverses M2 polarization of IL-10-producing tumor-associated macrophages (TAM) during efferocytosis induced by apoptotic cells. Mechanistically, Tα-1, which bound to phosphatidylserine on the surface of apoptotic tumor cells and was internalized by macrophages, triggered the activation of SH2-containing inositol 5′-phosphatase 1 (SHIP1) through the lysosomal toll-like receptor 7 (TLR7)/MyD88 pathway, subsequently resulting in dephosphorylation of efferocytosis-activated TBK1 and reduction of efferocytosis-induced IL-10. Tα-1 combined with epirubicin chemotherapy markedly suppressed tumor growth in an in vivo breast cancer model by reducing macrophage-derived IL-10 and enhancing the number and function of tumor-infiltrating CD4+ and CD8+ T cells. In conclusion, Tα-1 improved the curative effect of chemotherapy by reversing M2 polarization of efferocytosis-activated macrophages, suggesting that Tα-1 injection immediately after chemotherapy may contribute to highly synergistic anti-tumor effects in breast cancer patients.

Background and Aim: We have previously identified ubiquitinated proteins (UPs) from tumor cell lysates as a promising vaccine for cancer immunotherapy in different mouse tumor models. In this study, we aimed at developing a highly efficient therapeutic adjuvant built-in nanovaccine (α-Al 2 O 3-UPs) by a simple method, in which UPs from tumor cells could be efficiently and conveniently enriched by α-Al 2 O 3 nanoparticles covalently coupled with Vx3 proteins (α-Al 2 O 3-CONH-Vx3). Methods: The α-Al 2 O 3 nanoparticles were modified with 4-hydroxybenzoic acid followed by coupling with ubiquitin-binding protein Vx3. It was then used to enrich UPs from 4T1 cell lysate. The stability and the efficiency for the UPs enrichment of α-Al 2 O 3-CONH-Vx3 were examined. The ability of α-Al 2 O 3-UPs to activate DCs was examined in vitro subsequently. The splenocytes from the vaccinated mice were re-stimulated with inactivated tumor cells, and the IFN-γ secretion was detected by ELISA and flow cytometry. Moreover, the therapeutic efficacy of α-Al 2 O 3-UPs, alone and in combination with chemotherapy, was examined in 4T1 tumor-bearing mice. Results: Our results showed that α-Al 2 O 3-UPs were successfully synthesized and abundant UPs from tumor cell lysate were enriched by the new method. In vitro study showed that compared to the physical mixture of α-Al 2 O 3 nanoparticles and UPs (α-Al 2 O 3 +UPs), α-Al 2 O 3-UPs stimulation resulted in higher upregulations of CD80, CD86, MHC class I, and MHC class II on DCs, indicating the higher ability of DC activation. Moreover, α-Al 2 O 3-UPs elicited a more effective immune response in mice, demonstrated by higher IFN-γ secretion than α-Al 2 O 3 +UPs. Furthermore, α-Al 2 O 3-UPs also exhibited a more potent effect on tumor growth inhibition and survival prolongation in 4T1 tumor-bearing mice. Notably, when in combination with low dose chemotherapy, the anti-tumor effect was further enhanced, rather than using α-Al 2 O 3-UPs alone. Conclusion: This study presents an adjuvant built-in nanovaccine generated by a new simple method that can be potentially applied to cancer immunotherapy and lays the experimental foundation for future clinical application.

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Most breast cancer–related deaths are caused by metastasis in vital organs including the lungs. Development of supportive metastatic microenvironments, referred to as premetastatic niches (PMNs), in certain distant organs before arrival of metastatic cells, is critical in metastasis. However, the mechanisms of PMN formation are not fully clear. Here, we demonstrated that chemoattractant C–C motif chemokine ligand 2 (CCL2) could be stimulated by heat shock protein 60 (HSP60) on the surface of murine 4 T1 breast cancer cell–released LC3+ extracellular vesicles (LC3+ EVs) via the TLR2‐MyD88‐NF‐κB signal cascade in lung fibroblasts, which subsequently promoted lung PMN formation through recruiting monocytes and suppressing T cell function. Consistently, reduction of LC3+ EV release or HSP60 level or neutralization of CCL2 markedly attenuated PMN formation and lung metastasis. Furthermore, the number of circulating LC3+ EVs and HSP60 level on LC3+ EVs in the plasma of breast cancer patients were positively correlated with disease progression and lung metastasis, which might have potential value as biomarkers of lung metastasis in breast cancer patients (AUC = 0.898, 0.694, respectively). These findings illuminate a novel mechanism of PMN formation and might provide therapeutic targets for anti‐metastasis therapy for patients with breast cancer.

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