Size-advantage of monovalent nanobodies against the macrophage mannose receptor for deep tumor penetration and tumor-associated macrophage targeting

Rationale: Nanobodies (Nbs) have emerged as an elegant alternative to the use of conventional monoclonal antibodies in cancer therapy, but a detailed microscopic insight into the in vivo pharmacokinetics of different Nb formats in tumor-bearers is lacking. This is especially relevant for the recognition and targeting of pro-tumoral tumor-associated macrophages (TAMs), which may be located in less penetrable tumor regions.Methods: We employed anti-Macrophage Mannose Receptor (MMR) Nbs, in a monovalent (m) or bivalent (biv) format, to assess in vivo TAM targeting. Intravital and confocal microscopy were used to analyse the blood clearance rate and targeting kinetics of anti-MMR Nbs in tumor tissue, healthy muscle tissue and liver. Fluorescence Molecular Tomography was applied to confirm anti-MMR Nb accumulation in the primary tumor and in metastatic lesions.Results: Intravital microscopy demonstrated significant differences in the blood clearance rate and macrophage targeting kinetics of (m) and (biv)anti-MMR Nbs, both in tumoral and extra-tumoral tissue. Importantly, (m)anti-MMR Nbs are superior in reaching tissue macrophages, an advantage that is especially prominent in tumor tissue. The administration of a molar excess of unlabelled (biv)anti-MMR Nbs increased the (m)anti-MMR Nb bioavailability and impacted on its macrophage targeting kinetics, preventing their accumulation in extra-tumoral tissue (especially in the liver) but only partially influencing their interaction with TAMs. Finally, anti-MMR Nb administration not only allowed the visualization of TAMs in primary tumors, but also at a distant metastatic site.Conclusions: These data describe, for the first time, a microscopic analysis of (m) and (biv)anti-MMR Nb pharmacokinetics in tumor and healthy tissues. The concepts proposed in this study provide important knowledge for the future use of Nbs as diagnostic and therapeutic agents, especially for the targeting of tumor-infiltrating immune cells.Radiolog

Unleashing Tumour-Dendritic Cells to Fight Cancer by Tackling Their Three A’s: Abundance, Activation and Antigen-Delivery

Recent advances in cancer immunotherapy have mainly focused on re-activating T-cell responses against cancer cells. However, both priming and activation of effector T-cell responses against cancer-specific antigens require cross-talk with dendritic cells (DCs), which are responsible for the capturing, processing and presentation of tumour-(neo)antigens to T cells. DCs consequently constitute an essential target in efforts to generate therapeutic immunity against cancer. This review will discuss recent research that is unlocking the cancer-fighting potential of tumour-infiltrating DCs. First, the complexity of DCs in the tumour microenvironment regarding the different subsets and the difficulty of translating mouse data into equivalent human data will be briefly touched upon. Mainly, possible solutions to problems currently faced in DC-based cancer treatments will be discussed, including their infiltration into tumours, activation strategies, and antigen delivery methods. In this way, we hope to put together a broad picture of potential synergistic therapies that could be implemented to harness the full capacity of tumour-infiltrating DCs to stimulate anti-tumour immune responses in patients

Junctional adhesion molecule-A is dispensable for myeloid cell recruitment and diversification in the tumor microenvironment.

Junctional adhesion molecule-A (JAM-A), expressed on the surface of myeloid cells, is required for extravasation at sites of inflammation and may also modulate myeloid cell activation. Infiltration of myeloid cells is a common feature of tumors that drives disease progression, but the function of JAM-A in this phenomenon and its impact on tumor-infiltrating myeloid cells is little understood. Here we show that systemic cancer-associated inflammation in mice enhanced JAM-A expression selectively on circulating monocytes in an IL1β-dependent manner. Using myeloid-specific JAM-A-deficient mice, we found that JAM-A was dispensable for recruitment of monocytes and other myeloid cells to tumors, in contrast to its reported role in inflammation. Single-cell RNA sequencing revealed that loss of JAM-A did not influence the transcriptional reprogramming of myeloid cells in the tumor microenvironment. Overall, our results support the notion that cancer-associated inflammation can modulate the phenotype of circulating immune cells, and we demonstrate that tumors can bypass the requirement of JAM-A for myeloid cell recruitment and reprogramming

Opa3, a novel regulator of mitochondrial function, controls thermogenesis and abdominal fat mass in a mouse model for Costeff syndrome

The interrelationship between brown (BAT) and white (WAT) adipose tissue is emerging as an important factor in obesity, but the effect of impairing non-shivering thermogenesis in BAT on lipid storage in WAT remains unclear. To address this we have characterized the metabolic phenotype of a mouse model for Costeff syndrome, in which a point mutation in the mitochondrial membrane protein Opa3 impairs mitochondrial activity. Opa3L122P mice displayed an 80% reduction in insulin-like growth factor 1, postnatal growth retardation and hepatic steatosis. A 90% reduction in UCP1 expression in interscapular BAT was accompanied by a marked reduction in surface body temperature, with a 2.5-fold elevation in interscapular BAT mass and lipid storage. The sequestration of circulating lipid into BAT resulted in profound reductions in epididymal and retroperitoneal WAT mass, without affecting subcutaneous WAT. The histological appearance and intense mitochondrial staining in intra-abdominal WAT suggests significant “browning”, but with UCP1 expression in WAT of Opa3L122P mice only 62% of that in wild type littermates, any precursor differentiation does not appear to result in thermogenically active beige adipocytes. Thus, we have identified Opa3 as a novel regulator of lipid metabolism, coupling lipid uptake with lipid processing in liver and with thermogenesis in BAT. These findings indicate that skeletal and metabolic impairment in Costeff syndrome may be more significant than previously thought and that uncoupling lipid uptake from lipid metabolism in BAT may represent a novel approach to controlling WAT mass in obesity

IL1 beta promotes immune suppression in the tumor microenvironment independent of the inflammasome and gasdermin D

IL1 beta is a central mediator of inflammation. Secretion of IL1 beta typically requires proteolytic maturation by the inflammasome and formation of membrane pores by gasdermin D (GSDMD). Emerging evidence suggests an important role for IL1 beta in promoting cancer progression in patients, but the underlying mechanisms are ill-defined. Here, we have shown a key role for IL1 beta in driving tumor progression in two distinct mouse tumor models. Notably, activation of the inflammasome, caspase-8, as well as the pore-forming proteins GSDMD and mixed lineage kinase domain-like protein in the host were dispensable for the release of intratumoral bioactive IL1 beta. Inflammasome-independent IL1 beta release promoted systemic neutrophil expansion and fostered accumulation of T-cell-suppressive neutrophils in the tumor. Moreover, IL1 beta was essential for neutrophil infiltration triggered by antiangiogenic therapy, thereby contributing to treatment-induced immunosuppression. Deletion of IL1 beta allowed intratumoral accumulation of CD8(+) effectorT cells that subsequently activated tumor-associated macrophages. Depletion of either CD8(+) T cells or macrophages abolished tumor growth inhibition in IL1 beta-deficient mice, demonstrating a crucial role for CD8(+) T-cell-macrophage cross-talk in the antitumor immune response. Overall, these results support a tumor-romoting role for IL1 beta through establishing an immunosuppressive microenvironment and show that inflammasome activation is not essential for release of this cytokine in tumors

Mitochondrial–Nuclear Communication by Prohibitin Shuttling under Oxidative Stress

Mitochondrial-nuclear communication is critical to maintain mitochondrial activity under stress conditions. Adaptation of the mitochondria-nucleus network to changes in the intracellular oxidation and reduction milieu is critical for the survival of retinal and retinal pigment epithelial (RPE) cells, in relation to their high oxygen demand and rapid metabolism. However, the generation and transmittal of mitochondrial signal to the nucleus remains elusive. Previously, our in vivo study revealed that prohibitin is up-regulated in the retina but is down-regulated in RPE in the aging and diabetic model. In this study, the functional role of prohibitin in the retina and the RPE was studied using biochemical methods, including lipid binding assay, 2D gel electrophoresis, immunocytochemistry, Western blot, and knockdown approach. Protein depletion by siRNA characterized prohibitin as an anti-apoptotic molecule in mitochondria, while lipid binding assay demonstrated subcellular communications between mitochondria and the nucleus under oxidative stress. The changes of the expressions and localization of mitochondrial prohibitin triggered by reactive oxygen species are crucial for mitochondrial integrity. We propose that prohibitin shuttles between mitochondria and the nucleus as an anti-apoptotic molecule and a transcriptional regulator under stress environment in the retina and RPE