Silver-Based Terpyridine Complexes as Antitumor Agents.

Silver complexes bearing substituted terpyridine or tetra-2-pyridinylpyrazine ligands have been prepared and structurally characterised. The study of the anticancer properties of silver complexes with this type of ligands is scarce, despite the possibilities of combining the properties of the metal and the ability of the ligands for DNA binding. Here, the antiproliferative activity, stability, CT-DNA binding, and mechanism of cell death of these types of derivatives are studied. High cytotoxicity against different tumour cells was observed, and, more important, a great selectivity index has been detected between tumour cells and healthy lymphocytes T for some of these compounds. The CT-DNA interaction study has shown that these derivatives are able to interact with CT-DNA by moderate intercalation. Furthermore, cell death studies indicate that these derivatives promote the apoptosis by a mitochondrial pathway.

Cytofluorometric assessment of calreticulin exposure on CD38+ plasma cells from the human bone marrow.

Exposure of the endoplasmic reticulum chaperone calreticulin (CALR) on the surface of stressed and dying cells is paramount for their effective engulfment by professional antigen-presenting cells such as dendritic cells (DCs). Importantly, this is required (but not sufficient) for DCs to initiate an adaptive immune response that culminates with an effector phase as well as with the establishment of immunological memory. Conversely, the early exposure of phosphatidylserine (PS) on the outer layer of the plasma membrane is generally associated with the rapid engulfment of stressed and dying cells by tolerogenic macrophages. Supporting the clinical relevance of the CALR exposure pathway, the spontaneous or therapy-driven translocation of CALR to the surface of malignant cells, as well as intracellular biomarkers thereof, have been associated with improved disease outcome in patients affected by a variety of neoplasms, with the notable exception of multiple myeloma (MM). Here, we describe an optimized protocol for the flow cytometry-assisted quantification of surface-exposed CALR and PS on CD38+ plasma cells from the bone marrow of patients with MM. With some variations, we expect this method to be straightforwardly adaptable to the detection of CALR and PS on the surface of cancer cells isolated from patients with neoplasms other than MM.

Flow cytometry-assisted analysis of phenotypic maturation markers on an immortalized dendritic cell line.

Dendritic cells (DCs), and especially so conventional type I DCs (cDC1s), are fundamental regulators of anticancer immunity, largely reflecting their superior ability to engulf tumor-derived material and process it for cross-presentation on MHC Class I molecules to CD8+ cytotoxic T lymphocytes (CTLs). Thus, investigating key DC functions including (but not limited to) phagocytic capacity, expression of CTL-activating ligands on the cell surface, and cross-presentation efficacy is an important component of multiple immuno-oncology studies. Unfortunately, DCs are terminally differentiated cells, implying that they cannot be propagated indefinitely in vitro and hence must be generated ad hoc from circulating or bone marrow-derived precursors, which presents several limitations. Here, we propose a simple, cytofluorometric method to quantify phenotypic activation markers including CD80, CD86 and MHC class II molecules on the surface of a conditionally immortalized immature DC line that can be indefinitely propagated in vitro but also driven into maturation at will with a simple change in culture conditions. Upon appropriate scaling and automatization, this approach is compatible with high-throughput screening programs for the discovery of novel DC activators that do not suffer from batch variability and other limitations associated with the generation of fresh DCs.

Generation of transmitochondrial cybrids in cancer cells.

At odds with historical views suggesting that mitochondrial functions are largely dispensable for cancer cells, it is now clear that mitochondria have a major impact on malignant transformation, tumor progression and response to treatment. Mitochondria are indeed critical for neoplastic cells not only as an abundant source of ATP and other metabolic intermediates, but also as gatekeepers of apoptotic cell death and inflammation. Interestingly, while mitochondrial components are mostly encoded by nuclear genes, mitochondria contain a small, circular genome that codes for a few mitochondrial proteins, ribosomal RNAs and transfer RNAs. Here, we describe a straightforward method to generate transmitochondrial cybrids, i.e., cancer cells depleted of their mitochondrial DNA and reconstituted with intact mitochondria from another cellular source. Once established, transmitochondrial cybrids can be stably propagated and are valuable to dissect the specific impact of the mitochondrial genome on cancer cell functions.

Dinaciclib synergizes with BH3 mimetics targeting BCL-2 and BCL-XL in multiple myeloma cell lines partially dependent on MCL-1 and in plasma cells from patients.

A better understanding of multiple myeloma (MM) biology has led to the development of novel therapies. However, MM is still an incurable disease and new pharmacological strategies are needed. Dinaciclib, a multiple cyclin-dependent kinase (CDK) inhibitor, which inhibits CDK1, 2, 5 and 9, displays significant antimyeloma activity as found in phase II clinical trials. In this study, we have explored the mechanism of dinaciclib-induced death and evaluated its enhancement by different BH3 mimetics in MM cell lines as well as in plasma cells from MM patients. Our results indicate a synergistic effect of dinaciclib-based combinations with B-cell lymphoma 2 or B-cell lymphoma extra-large inhibitors, especially in MM cell lines with partial dependence on myeloid cell leukemia sequence 1 (MCL-1). Simultaneous treatment with dinaciclib and BH3 mimetics ABT-199 or A-1155463 additionally showed a synergistic effect in plasma cells from MM patients, ex vivo. Altered MM cytogenetics did not affect dinaciclib response ex vivo, alone or in combined treatment, suggesting that these combinations could be a suitable therapeutic option for patients bearing cytogenetic alterations and poor prognosis. This work also opens the possibility to explore cyclin-dependent kinase 9 inhibition as a targeted therapy in MM patients overexpressing or with high dependence on MCL-1.

Toll-like receptors: New targets for multiple myeloma treatment?

Despite recent biomedical improvements in treating multiple myeloma, this disease still remains incurable. Toll-like receptors (TLRs) are key immune receptors that recognize conserved molecular patterns expressed by pathogens and damaged cells. Activation of TLRs can induce several effects including inflammatory responses, modulation of cell cycle, apoptosis, or regulation of cell metabolism. In multiple myeloma there is a dysregulated signalling of TLRs due to an abnormal presence of certain pathogens and release of molecules from damaged cells. Thus, TLRs could be critical players for tumour microenvironment and multiple myeloma progression. This haematological malignancy is characterized by a high percentage of recurrences, where many patients can develop residual drug-resistant malignant cells. Strategic targeting of TLRs might result in novel therapeutic combinations that improve the response to current treatments, reducing relapses. This review examines the potential of TLRs as targets for the treatment of multiple myeloma, making a particular emphasis on their therapeutic applications.

Ecto-calreticulin expression in multiple myeloma correlates with a failed anti-tumoral immune response and bad prognosis.

Immunogenic cell death (ICD) has been proposed to be a crucial process for antitumor immunosurveillance. ICD is characterized by the exposure and emission of Damage Associated Molecular Patterns (DAMP), including calreticulin (CRT). A positive correlation between CRT exposure or total expression and improved anticancer immunosurveillance has been found in certain cancers, usually accompanied by favorable patient prognosis. In the present study, we sought to evaluate CRT levels in the plasma membrane of CD38+ bone marrow mononuclear cells (BMMCs) isolated from 71 patients with varying degrees of multiple myeloma (MM) disease and examine the possible relationship between basal CRT exposure and the bone marrow immune microenvironment, as well as its connection with different clinical markers. Data show that increased levels of cell surface-CRT were associated with more aggressive clinical features and with worse clinical prognosis in MM. High CRT expression in MM cells was associated with increased infiltration of NK cells, CD8+ T lymphocytes and dendritic cells (DC), indicative of an active anti-tumoral immune response, but also with a significantly higher presence of immunosuppressive Treg cells and increased expression of PD-L1 in myeloma cells.

Efficient encapsulation of theranostic nanoparticles in cell-derived exosomes: leveraging the exosomal biogenesis pathway to obtain hollow gold nanoparticle-hybrids.

Exosomes can be considered natural targeted delivery systems able to carry exogenous payloads, drugs or theranostic nanoparticles (NPs). This work aims to combine the therapeutic capabilities of hollow gold nanoparticles (HGNs) with the unique tumor targeting properties provided by exosomes. Here, we tested different methods to encapsulate HGNs (capable of absorbing light in the NIR region for selective thermal ablation) into murine melanoma cells derived exosomes (B16-F10-exos), including electroporation, passive loading by diffusion, thermal shock, sonication and saponin-assisted loading. These methods gave less than satisfactory results: although internalization of relatively large NPs into B16-F10-exos was achieved by almost all the physicochemical methods tested, only about 15% of the exosomes were loaded with NPs and several of those processes had a negative effect regarding the morphology and integrity of the loaded exosomes. In a different approach, B16-F10 cells were pre-incubated with PEGylated HGNs (PEG-HGNs) in an attempt to incorporate the NPs into the exosomal biogenesis pathway. The results were highly successful: exosomes recovered from the supernatant of the cell culture showed up to 50% of HGNs internalization. The obtained hybrid HGN-exosome vectors were characterized with a battery of techniques to make sure that internalization of HGNs did not affect exosome characteristics compared with other strategies. PEG-HGNs were released through the endosomal-exosome biogenesis pathway confirming that the isolated vesicles were exosomes.