M2-polarized tumor-associated macrophages (TAM) play a critical role in cancer invasion and metastasis. Here, we report that M2 macrophages enhanced metastasis of K7M2 WT osteosarcoma cells to the lungs in mice, thus establishing M2 TAMs as a therapeutic target for blocking osteosarcoma metastasis. We found that retinoic acid (ATRA) inhibited osteosarcoma metastasis via inhibiting the M2 polarization of TAMs. ATRA suppressed IL13- or IL4-induced M2-type macrophages, and then inhibited migration of osteosarcoma cells as promoted by M2-type macrophages ATRA reduced the number of pulmonary metastatic nodes of osteosarcoma and decreased expression of M2-type macrophages in metastatic nodes both in intravenous injection and orthotopic transplantation models. ATRA's effect was independent of conventional STAT3/6 or C/EBPβ signaling, which regulate M2-like polarization of macrophages. Quantitative genomic and functional analyses revealed that MMP12, a macrophage-secreted elastase, was elevated in IL13-skewed TAM polarization, whereas ATRA treatment downregulated IL13-induced secretion of MMP12. This downregulation correlates with the antimetastasis effect of ATRA. Our results show the role of TAM polarization in osteosarcoma metastasis, identify a therapeutic opportunity for antimetastasis treatment, and indicate ATRA treatment as an approach for preventing osteosarcoma metastasis via M2-type polarization intervention. .

SummaryAdriamycin-based combination chemotherapy is the standard first-line treatment for osteosarcoma, but tumor recurrence and metastasis occurs in most cases. Recent evidence suggests that microenvironmental stress such as chemotherapy can lead to the enrichment of cancer stem cells (CSCs), which result in cancer metastasis, recurrence, and drug resistance. However, the exact mechanisms underlying this phenomenon and how to target CSCs are still open questions. Herein, we report that Adriamycin treatment induces a stem-like phenotype and promotes metastatic potential in osteosarcoma cells through upregulating KLF4. KLF4 knockdown blocks Adriamycin-induced stemness phenotype and metastasis capacity. We further screen that statins remarkably reverse Adriamycin-induced CSC properties and metastasis by downregulating KLF4. Most strikingly, simvastatin severely impaired Adriamycin-enhanced tumorigenesis of KHOS/NP cells in vivo. These data suggest that Adriamycin-based chemotherapeutics may simulate CSCs through activation of KLF4 signaling and that selective inhibition of KLF4 with statins should be considered in the development of osteosarcoma therapeutics.

CD8 + T cell longevity regulated by metabolic activity plays important roles in cancer immunotherapy. Although in vitro polarized, transferred IL-9-secreting CD8 + Tc9 cells exert greater persistence and antitumor efficacy than Tc1/CTL cells, the underlying mechanism remains unclear. Here, we show that tumor-infiltrating Tc9 cells display significantly lower lipid peroxidation than Tc1 cells in several mouse models, which is strongly correlated with their persistence. Using RNA-sequence and functional validation, we found that Tc9 cells exhibited unique lipid metabolic programs. Tc9 cell-derived IL-9 activated STAT3, upregulated fatty acid oxidation and mitochondrial activity, and rendered Tc9 cells with reduced lipid peroxidation and resistant to tumor or ROS induced ferroptosis in TME. IL-9 signal deficiency, inhibiting STAT3 or fatty acid oxidation increased lipid peroxidation and ferroptosis of Tc9 cells, resulting in impaired longevity and antitumor ability. Similarly, human Tc9 cells also possessed lower lipid peroxidation than Tc1 cells and tumor-infiltrating CD8 + T cells expressed lower IL9 and higher lipid peroxidation-and ferroptosis-related genes than circulating CD8 + T cells in melanoma patients. This study indicates that lipid peroxidation regulates Tc9-cell longevity and antitumor effects via IL-9/STAT3/fatty acid oxidation pathway and regulating T-cell lipid peroxidation can be used to enhance T-cell based immunotherapy in human cancer.

Multiple myeloma (MM) remains largely incurable despite significant advances in bio- and chemotherapy. The major problem in MM management is development of drug resistance. Macrophage migration inhibitory factor (MIF) expression was significantly higher in purified MM cells from relapsed patients than those with sustained response, and high MIF MM patients had significantly shorter progression-free survival (PFS) and overall survival (OS). MM cell lines also express high levels of MIF, and knocking out MIF made them more sensitive to proteasome inhibitor (PI) induced apoptosis not observed with other chemotherapy drugs. Mechanistic studies showed that MIF protects MM cells from PI-induced apoptosis by maintaining mitochondrial function via suppression of superoxide production in response to PIs. Specifically, MIF, in the form of a homotrimer, acts as a chaperone for superoxide dismutase 1 (SOD1) to suppress PI-induced SOD1 misfolding and maintain SOD1 activity. MIF inhibitor 4-IPP and homotrimer disrupter ebselen, which do not kill MM cells, enhanced PI-induced SOD1 misfolding and loss of function, resulting in significantly more cell death in both cell lines and primary MM cells. More importantly, inhibiting MIF activity in vivo displayed synergistic antitumor activity with PIs and re-sensitized PI-resistant MM cells to treatment. In support of these findings, gene-profiling data showed a significantly negative correlation between MIF and SOD1 expression and response to PI treatment in MM patients. Hence, this study reveals that MIF plays a crucial role in MM sensitivity to PIs, and suggests that targeting MIF may be a promising strategy to (re)sensitize MM to the treatment.

Osteosarcoma is the most common bone cancer, and chemotherapy is currently indispensable for its treatment. Adriamycin has been claimed to be the most effective agent for osteosarcoma, however, the outcome of adriamycin chemotherapy remains unsatisfactory. Here, we reported a potent combination therapy that bortezomib, a proteasome inhibitor, enhances adriamycin-induced apoptosis to eliminate osteosarcoma cells and we revealed that the activation of p-eIF2α/ATF4/CHOP axis is the underlying associated mechanisms. First, we observed that bortezomib enhances adriamycin-mediated inhibition of cell proliferation and enhances the apoptosis in osteosarcoma cell lines. Moreover, this drug combination produced more potent tumor-growth inhibitory effects in human osteosarcoma cell line KHOS/NP xenografts. Our study showed that reactive oxygen species (ROS) plays an important role in apoptosis induced by adriamycin plus bortezomib, whereas ROS scavenger NAC could almost completely block the apoptosis induced by the combination treatment. Meanwhile, p-eIF2α is remarkably elevated in the combination group. As a result, ATF4 exhibits strong activation which consequently induces the activation of CHOP and leads to the cell death. Finally, 13 primary osteosarcoma cells demonstrated potent response to the combination treatment. In a human osteosarcoma patient-derived xenograft (PDX) model, our finding suggests that when combined with bortezomib, a relatively low dose of adriamycin produced more potent tumor-growth inhibitory effects without increased toxicity. Thus, our findings not only provide a promising combination strategy to overcome osteosarcoma but also shed new light on the strategy of combining increased ROS and inhibited proteasome to open up new opportunities for the clinical development of chemotherapy regimens.