Acid microenvironment promotes cell survival of human bone sarcoma through the activation of cIAP proteins and NF-κB pathway

Extracellular acidification is a very common cause of stress in tumor microenvironment and of Darwinian pressure. In acid areas of the tumor, most cancer cells are-albeit slowly proliferating-more resistant to cell death than those in well-perfused regions. Tumor acidosis can directly regulate the expression of pro-survival proteins since a low extracellular pH activates the caspase-dependent cell death machinery. This mechanism has never been explored in bone sarcomas. We cultured osteosarcoma and Ewing sarcoma cells under low pH (pH 6.5), and we performed deep-sequencing and protein analysis. Both in in vitro and in vivo models, acidification activity enhanced tumor cells survival. However, we did not observe any change in ERK1 phosphorylation. On the contrary, both at the mRNA and protein level, we found a significant induction of TRAF adaptor proteins and of cIAP proteins (BIRC2 and/or BIRC3). As a consequence, the downstream nuclear transcription factor kappa B (NF-κB) survival pathway was increased. Furthermore, the treatment with the cIAP inhibitor LCL161 reverted the protection from apoptosis under low pH. In vitro results were confirmed both in Ewing sarcoma xenograft and in osteosarcoma patients, since the analysis of tumor tissues demonstrated that the levels of expression of TRAF1 or NF-κB1 significantly correlate with the level of expression of the vacuolar ATPase (V-ATPase), the most important proton pump in eukaryotes. Moreover, in the tissue sections of xenograft model, the nuclear translocation of RelB, a key subunit of the NF-κB transcriptional complex, localized in the tumor region that also corresponded to the acid microenvironment associated with the highest levels of expression of LAMP2 and V-ATPase, in the internal area of the tumor, as revealed by immunohistochemistry. Our data confirm that tumor acid microenvironment activates a stress-regulated switch to promote cell survival of bone sarcoma, and support the hypothesis that this mechanism is mediated by the recruitment of TRAF/cIAP complexes. Altogether, these results suggest that TRAF/cIAP can be considered as a target for anti-cancer therapies

Deepening the knowledge of ros1 rearrangements in non-small cell lung cancer: Diagnosis, treatment, resistance and concomitant alterations

ROS proto-oncogene 1 (ROS1) rearrangements are reported in about 1–2% of non-squamous non-small-cell lung cancer (NSCLC). After efficacy of crizotinib was demonstrated, identification of ROS1 translocations in advanced disease became fundamental to give patients the chance of specific and effective treatment. Different methods are available for detection of rearrangements, and probably the real prevalence of ROS1 rearrangements is higher than that reported in literature, as our capacity to detect gene rearrangements is improving. In particular, with next generation sequencing (NGS) techniques, we are currently able to assess multiple genes simultaneously with increasing sensitivity. This is leading to overcome the “single oncogenic driver” paradigm, and in the very near future, the co-existence of multiple drivers will probably emerge more frequently and represent a therapeutic issue. Since recently, crizotinib has been the only available therapy, but today, many other tyrosine kinase inhibitors (TKI) are emerging and seem promising both in first and subsequent lines of treatment. Indeed, novel inhibitors are also able to overcome resistance mutations to crizotinib, hypothesizing a possible sequential strategy also in ROS1-rearranged disease. In this review, we will focus on ROS1 rearrangements, dealing with diagnostic aspects, new therapeutic options, resistance issues and the coexistence of ROS1 translocations with other molecular alterations

A 3D Platform to Investigate Dynamic Cell-to-Cell Interactions Between Tumor Cells and Mesenchymal Progenitors

We here investigated the dynamic cell-to-cell interactions between tumor and mesenchymal stromal/stem cells (MSCs) by the novel VITVOⓇ 3D bioreactor that was customized to develop in vivo-like metastatic nodules of Ewing’s sarcoma (ES). MSCs are known to contribute to tumor microenvironment as cancer associated fibroblast (CAF) precursors and, for this reason, they have also been used as anti-cancer tools. Using dynamic conditions, the process of tissue colonization and formation of metastatic niches was recreated through tumor cell migration aiming to mimic ES development in patients. ES is an aggressive tumor representing the second most common malignant bone cancer in children and young adults. An urgent and unmet need exists for the development of novel treatment strategies to improve the outcomes of metastatic ES. The tumor-tropic ability of MSCs offers an alternative approach, in which these cells can be used as vehicles for the delivery of antitumor molecules, such as the proapoptotic TNF-related apoptosis inducing ligand (TRAIL). However, the therapeutic targeting of metastases remains challenging and the interaction occurring between tumor cells and MSCs has not yet been deeply investigated. Setting up in vitro and in vivo models to study this interaction is a prerequisite for novel approaches where MSCs affinity for tumor is optimized to ultimately increase their therapeutic efficacy. Here, VITVOⓇ integrating a customized scaffold with an increased inter-fiber distance (VITVO50) was used to develop a dynamic model where MSCs and tumor nodules were evaluated under flow conditions. Colonization and interaction between cell populations were explored by droplet digital PCR (ddPCR). VITVO50 findings were then applied in vivo. An ES metastatic model was established in NSG mice and biodistribution of TRAIL-expressing MSCs in mice organs affected by metastases was investigated using a 4-plex ddPCR assay. VITVOⓇ proved to be an easy handling and versatile bioreactor to develop in vivo-like tumor nodules and investigate dynamic cell-to-cell interactions with MSCs. The proposed fluidic system promises to facilitate the understanding of tumor-stroma interaction for the development of novel tumor targeting strategies, simplifying the analysis of in vivo data, and ultimately accelerating the progress towards the early clinical phase

Targeting GD2-positive glioblastoma by chimeric antigen receptor empowered mesenchymal progenitors

Tumor targeting by genetically modified mesenchymal stromal/stem cells (MSCs) carrying anti-cancer molecules represents a promising cell-based strategy. We previously showed that the pro-apoptotic agent tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can be successfully delivered by MSCs to cancer sites. While the interaction between TRAIL and its receptors is clear, more obscure is the way in which MSCs can selectively target tumors and their antigens. Several neuroectoderm-derived neoplasms, including glioblastoma (GBM), sarcomas, and neuroblastoma, express high levels of the tumor-associated antigen GD2. We have already challenged this cell surface disialoganglioside by a chimeric antigen receptor (CAR)-T cell approach against neuroblastoma. With the intent to maximize the therapeutic profile of MSCs delivering TRAIL, we here originally developed a bi-functional strategy where TRAIL is delivered by MSCs that are also gene modified with the truncated form of the anti-GD2 CAR (GD2 tCAR) to mediate an immunoselective recognition of GD2-positive tumors. These bi-functional MSCs expressed high levels of TRAIL and GD2 tCAR associated with a robust anti-tumor activity against GD2-positive GBM cells. Most importantly, the anti-cancer action was reinforced by the enhanced targeting potential of such bi-functional cells. Collectively, our results suggest that a truncated anti-GD2 CAR might be a powerful new tool to redirect MSCs carrying TRAIL against GD2-expressing tumors. This affinity-based dual targeting holds the promise to combine site-specific and prolonged retention of MSCs in GD2-expressing tumors, thereby providing a more effective delivery of TRAIL for still incurable cancers

IGF-1-mediated osteoblastic niche expansion enhances long-term hematopoietic stem cell engraftment after murine bone marrow transplantation

The efficiency of hematopoietic stem cell (HSC) engraftment after bone marrow (BM) transplantation depends largely on the capacity of the marrow microenvironment to accept the transplanted cells. While radioablation of BM damages osteoblastic stem cell niches, little is known about their restoration and mechanisms governing their receptivity to engraft transplanted HSCs. We previously reported rapid restoration and profound expansion of the marrow endosteal microenvironment in response to marrow radioablation. Here, we show that this reorganization represents proliferation of mature endosteal osteoblasts which seem to arise from a small subset of high-proliferative, relatively radio-resistant endosteal cells. Multiple layers of osteoblasts form along the endosteal surface within 48 hours after total-body irradiation, concomitant with a peak in marrow cytokine expression. This niche reorganization fosters homing of the transplanted hematopoietic cells to the host marrow space and engraftment of long-term (LT)-HSC. Inhibition of insulin-like growth factor (IGF)-1-receptor tyrosine kinase signaling abrogates endosteal osteoblast proliferation and donor HSC engraftment, suggesting that the cytokine IGF-1 is a crucial mediator of endosteal niche reorganization and consequently donor HSC engraftment. Further understanding of this novel mechanism of IGF-1-dependent osteoblastic niche expansion and HSC engraftment may yield clinical applications for improving engraftment efficiency after clinical HSC transplantation.The efficiency of hematopoietic stem cell (HSC) engraft-ment after bone marrow (BM) transplantation depends largely on the capacity of the marrow microenvironment to accept the transplanted cells. While radioablation of BM damages osteoblastic stem cell niches, little is known about their restoration and mechanisms governing their receptivity to engraft transplanted HSCs. We previously reported rapid restoration and profound expansion of the marrow endosteal microenvironment in response to marrow radioablation. Here, we show that this reorganization represents proliferation of mature endosteal osteoblasts which seem to arise from a small subset of high-proliferative, relatively radio-resistant endosteal cells. Multiple layers of osteoblasts form along the endosteal surface within 48 hours after total body irradiation, concomitant with a peak in marrow cytokine expression. This niche reorganization fosters homing of the transplanted hematopoietic cells to the host marrow space and engraft-ment of long-term-HSC. Inhibition of insulin-like growth factor (IGF)-1-receptor tyrosine kinase signaling abrogates endosteal osteoblast proliferation and donor HSC engraft-ment, suggesting that the cytokine IGF-1 is a crucial mediator of endosteal niche reorganization and consequently donor HSC engraftment. Further understanding of this novel mechanism of IGF-1-dependent osteoblastic niche expansion and HSC engraftment may yield clinical applications for improving engraftment efficiency after clinical HSC transplantation. © AlphaMed Press

WISP-2 expression induced by Teriparatide treatment affects in vitro osteoblast differentiation and improves in vivo osteogenesis

The Osteocyte, recognized as a major orchestrator of osteoblast and osteoclast activity, is the most important key player during bone remodeling processes. Imbalances occurring during bone remodeling, caused by hormone perturbations or by mechanical loading alterations, can induce bone pathologies such as osteoporosis. Recently, the active fraction of parathormone, PTH (1-34) or Teriparatide (TPTD), was chosen as election treatment for osteoporosis. The effect of such therapy is dependent on the temporal manner of administration. The molecular reasons why the type of administration regimen is so critical for the fate of bone remodeling are numerous and not yet well known. Our study attempts to analyze diverse signaling pathways directly activated in osteocytes upon TPTD treatment. By means of gene array analysis, we found many molecules upregulated or downregulated in osteocytes. Later, we paid attention to Wisp-2, a protein involved in the Wnt pathway, that is secreted by MLO-Y4 cells and increases upon TPTD treatment and that is able to positively influence the early phases of osteogenic differentiation. We also confirmed the pro osteogenic property of Wisp-2 during mesenchymal stem cell differentiation into the preliminary osteoblast phenotype. The same results were confirmed with an in vivo approach confirming a remarkable Wisp-2 expression in metaphyseal trabecular bone. These results highlighted the anabolic roles unrolled by osteocytes in controlling the action of neighboring cells, suggesting that the perturbation of certain signaling cascades, such as the Wnt pathway, is crucial for the positive regulation of bone formation

Resistance to neoplastic transformation of ex-vivo expanded human mesenchymal stromal cells after exposure to supramaximal physical and chemical stress

The risk of malignant transformation of ex-vivo expanded human mesenchymal stromal cells (huMSCs) has been debated in the last years; however, the biosafety of these cells after exposure to supramaximal physical and chemical stress has never been systematically investigated. We established an experimental in vitro model to induce supramaximal physical (ionizing radiation, IR) and chemical (starvation) stress on ex-vivo expanded bone marrow (BM)-derived huMSCs and investigated their propensity to undergo malignant transformation. To this aim, we examined MSC morphology, proliferative capacity, immune-phenotype, differentiation potential, immunomodulatory properties and genetic profile before and after stressor exposure. Furthermore, we investigated the cellular mechanisms underlying MSC response to stress. MSCs were isolated from 20 healthy BM donors and expanded in culture medium supplemented with 5% platelet lysate (PL) up to passage 2 (P2). At this stage, MSCs were exposed first to escalating doses of IR (30, 100, 200 Gy) and then to starvation culture conditions (1% PL). With escalating doses of radiation, MSCs lost their typical spindle-shaped morphology, their growth rate markedly decreased and eventually stopped (at P4-P6) by reaching early senescence. Irradiated and starved MSCs maintained their typical immune-phenotype, ability to differentiate into adipocytes/osteoblasts and to inhibit mitogen-induced T-cell proliferation. The study of the genetic profile of irradiated/ starved MSCs did not show any alteration. While the induction of supramaximal stress triggered production of ROS and activation of DNA damage response pathway via multiple mechanisms, our data indicate that irradiated/starved MSCs, although presenting altered morphology/growth rate, do not display increased propensity for malignant transformation

Understanding Tumor-Stroma Interplays for Targeted Therapies by Armed Mesenchymal Stromal Progenitors: The Mesenkillers.

Tumor represents a complex structure containing malignant cells strictly coupled with a large variety of surroundingcells constituting the tumor stroma (TS). In recent years, the importance of TS for cancer initiation, development,local invasion and metastases became increasingly clear allowing the identification of TS as one of the possibleways to indirectly target tumors. Inside the heterogeneous stromal cell population, tumor associated fibroblasts(TAF) play a crucial role providing both functional and supportive environments. During both tumor and stroma development,several findings suggest that TAF could be recruited from different sources such as locally derived host fibroblasts,via epithelial/endothelial mesenchymal transitions or from circulating pools of fibroblasts deriving form mesenchymalprogenitors, namely mesenchymal stem/stromal cells (MSC). These insights prompted scientists to identifymultimodal approaches to target TS by biomolecules, monoclonal antibodies and, more recently, via cell basedstrategies. These latter appear extremely promising, although associated with still debated and unclear findings. Thisreview discusses on crosstalk between cancers and their stroma, dissecting specific tumor types, such as sarcoma,pancreatic and breast carcinoma where stroma plays distinct paradigmatic roles. The recognition of these distinctstromal functions may help in planning effective and safer approaches aimed either to eradicate or to substitute TSby novel compounds and/or MSC having specific killing activitie

Human Adipose Mesenchymal Stromal/Stem Cells Improve Fat Transplantation Performance

The resorption rate of autologous fat transfer (AFT) is 40–60% of the implanted tissue, requiring new surgical strategies for tissue reconstruction. We previously demonstrated in a rabbit model that AFT may be empowered by adipose-derived mesenchymal stromal/stem cells (AD-MSCs), which improve graft persistence by exerting proangiogenic/anti-inflammatory effects. However, their fate after implantation requires more investigation. We report a xenograft model of adipose tissue engineering in which NOD/SCID mice underwent AFT with/without human autologous AD-MSCs and were monitored for 180 days (d). The effect of AD-MSCs on AFT grafting was also monitored by evaluating the expression of CD31 and F4/80 markers. Green fluorescent protein-positive AD-MSCs (AD-MSC-GFP) were detected in fibroblastoid cells 7 days after transplantation and in mature adipocytes at 60 days, indicating both persistence and differentiation of the implanted cells. This evidence also correlated with the persistence of a higher graft weight in AFT-AD-MSC compared to AFT alone treated mice. An observation up to 180 d revealed a lower resorption rate and reduced lipidic cyst formation in the AFT-AD-MSC group, suggesting a long-term action of AD-MSCs in support of AFT performance and an anti-inflammatory/proangiogenic activity. Together, these data indicate the protective role of adipose progenitors in autologous AFT tissue resorption