Harnessing the cross-talk between tumor cells and tumor-associated macrophages with a nano-drug for modulation of glioblastoma immune microenvironment.

Glioblastoma (GBM) is the most frequent and malignant brain tumor with a high mortality rate. The presence of a large population of macrophages (Mφ) in the tumor microenvironment is a prominent feature of GBM and these so-called tumor-associated Mφ (TAM) closely interact with the GBM cells to promote the survival, progression and therapy resistance of the GBM. Various therapeutic strategies have been devised either targeting the GBM cells or the TAM but few have addressed the cross-talks between the two cell populations. The present study was carried out to explore the possibility of exploiting the cross-talks between the GBM cells (GC) and TAM for modulation of the GBM microenvironment through using Nano-DOX, a drug composite based on nanodiamonds bearing doxorubicin. In the in vitro work on human cell models, Nano-DOX-loaded TAM were first shown to be viable and able to infiltrate three-dimensional GC spheroids and release cargo drug therein. GC were then demonstrated to encourage Nano-DOX-loaded TAM to unload Nano-DOX back into GC which consequently emitted damage-associated molecular patterns (DAMPs) that are powerful immunostimulatory agents as well as indicators of cell damage. Nano-DOX was next proven to be a more potent inducer of GC DAMPs emission than doxorubicin. As a result, Nano-DOX-damaged GC exhibited an enhanced ability to attract both TAM and Nano-DOX-loaded TAM. Most remarkably, Nano-DOX-damaged GC reprogrammed the TAM from a pro-GBM phenotype to an anti-GBM phenotype that suppressed GC growth. Finally, the in vivo relevance of the in vitro findings was tested in animal study. Mice bearing orthotopic human GBM xenografts were intravenously injected with Nano-DOX-loaded mouse TAM which were found releasing drug in the GBM xenografts 24h after injection. GC damage was evidenced by the induction of DAMPs emission within the xenografts and a shift of TAM phenotype was detected as well. Taken together, our results demonstrate a novel way with therapeutic potential to harness the cross-talk between GBM cells and TAM for modulation of the tumor immune microenvironment.

Li TF ，Li K ，Wang C ，Liu X ，Wen Y ，Xu YH ，Zhang Q ，Zhao QY ，Shao M ，Li YZ ，Han M ，Komatsu N ，Zhao L ，Chen X ... -  《-》

Dendritic cell-mediated delivery of doxorubicin-polyglycerol-nanodiamond composites elicits enhanced anti-cancer immune response in glioblastoma.

Glioblastoma (GBM) is the deadliest and most common type of primary brain tumor in adults with a grim prognosis despite multimodal treatments. Dendritic cell (DC)-based immunotherapy has emerged as a promising therapeutic modality for GBM, whose efficacy is nonetheless fundamentally undermined by GBM-induced immunosuppression. Inducing emission of damage associated molecular patterns (DAMPs) is a highly effective strategy to subvert tumor-associated immunosuppression. The present work was carried out to explore the idea of subverting the GBM immunosuppressive microenvironment through DC-mediated delivery of doxorubicin-polyglycerol-nanodiamond composites (Nano-DOX), a potent DAMPs inducer demonstrated by our previous study, and thereby eliciting enhanced DC-driven anti-GBM immune response. In the in-vitro work on human cell models, Nano-DOX-loaded DC were shown to be functionally viable and release cargo drug to co-cultured GBM cells (GC). Nano-DOX-treated GC displayed not only profuse DAMPs emission but also antigen release. Enhanced activation and acquisition and presentation of GC-derived antigen were then demonstrated in DC in co-culture with GC and Nano-DOX. Consistently, co-culture with GC and Nano-DOX also activated mouse bone marrow-derived DC (mDC) which in turn stimulated mouse spleen-derived lymphocytes which ultimately suppressed co-cultured GC. Next, athymic mice bearing orthotopic human GBM xenografts were intravenously injected with Nano-DOX-loaded mDC and, 48 h later, spleen-derived lymphocytes. The presence of Nano-DOX, DAMPs emission and enhanced infiltration and activation of mDC and lymphocytes were detected in the GBM xenografts. Taken together, our results demonstrate the efficacy of DC-mediated delivery of Nano-DOX to stimulate GC immunogenicity and elicit anti-cancer immune response in the GBM. By this work, we present a novel approach with great application potential to subverting the GBM immunosuppressive microenvironment and to anti-GBM immunotherapy. Investigation has also been conducted probing the mechanisms by which Nano-DOX stimulates GC immunogenicity, which is described in a follow-up paper.

Li TF ，Li K ，Zhang Q ，Wang C ，Yue Y ，Chen Z ，Yuan SJ ，Liu X ，Wen Y ，Han M ，Komatsu N ，Xu YH ，Zhao L ，Chen X ... -  《-》

Doxorubicin-polyglycerol-nanodiamond composites stimulate glioblastoma cell immunogenicity through activation of autophagy.

Immunosuppression is a salient feature of GBM associated with the disease's grim prognosis and the limited success of anti-GBM immunotherapy. Stimulating immunogenicity of the GBM cells (GC) is a promising approach to subverting the GBM-associated immunosuppression. We had previously devised a drug composite based on polyglycerol-functionalized nanodiamonds bearing doxorubicin (Nano-DOX) and demonstrated that Nano-DOX effectively modulated GBM's immunosuppressive microenvironment through stimulating the immunogenicity of GC and initiated anti-GBM immune responses. The present study now explored the mechanism of Nano-DOX's immunostimulatory action. Nano-DOX was found to induce autophagy rather than apoptosis in GC and stimulated GC to emit antigens and damage-associated molecular patterns (DAMPs) that are potent adjuvants, which resulted in enhanced activation of dendritic cells (DC). Heightened autophagosome release was observed in Nano-DOX-treated GC but was shown not to be a major channel of antigen donation. Blocking autophagy in GC not only reduced Nano-DOX-stimulated GC antigen donation and DAMPs emission, but also efficiently attenuated DC activation stimulated by Nano-DOX-treated GC. Taken together, these findings suggest that activation of autophagy is a central mechanism whereby Nano-DOX stimulates GC's immunogenicity. Our work provides new insight on how nanotechnology can be applied to therapeutically modulate the GBM immune microenvironment by harnessing autophagy in the cancer cells. STATEMENT OF SIGNIFICANCE: Immunosuppression is a salient feature of GBM associated with the grim prognosis of the disease and the limited success of anti-GBM immunotherapy. We demonstrated that Doxorubicin-polyglycerol-nanodiamond composites could activate autophagy in GBM cells and thereby stimulate the immunogenecity of GBM cells. This discovery 1, sheds new light on how nanotechnology could be applied to therapeutically modulate the tumor immune microenvironment, and 2, provides a powerful tool for subverting the GBM's immunosuppressive microenvironment, which has great therapeutic potential for the treatment of GBM.

Li TF ，Xu YH ，Li K ，Wang C ，Liu X ，Yue Y ，Chen Z ，Yuan SJ ，Wen Y ，Zhang Q ，Han M ，Komatsu N ，Zhao L ，Chen X ... -  《-》

Monocyte-mediated chemotherapy drug delivery in glioblastoma.

Wang C ，Li K ，Li T ，Chen Z ，Wen Y ，Liu X ，Jia X ，Zhang Y ，Xu Y ，Han M ，Komatsu N ，Zhao L ，Chen X ... -  《-》

Doxorubicin conjugated with nanodiamonds and in free form commit glioblastoma cells to heterodromous fates.

Chen Z ，Wang C ，Li TF ，Li K ，Yue Y ，Liu X ，Xu HZ ，Wen Y ，Zhang Q ，Han M ，Komatsu N ，Xu YH ，Zhao L ，Chen X ... -  《-》