Multiple myeloma (MM) is a hematologic cancer characterized by clonal proliferation of plasma cells in the bone marrow (BM). The progression, from the early stages of the disease as monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM) to MM and occasionally extramedullary disease, is drastically affected by the tumor microenvironment (TME). Soluble factors and direct cell–cell interactions regulate MM plasma cell trafficking and homing to the BM niche. Mesenchymal stromal cells, osteoclasts, osteoblasts, myeloid and lymphoid cells present in the BM create a unique milieu that favors MM plasma cell immune evasion and promotes disease progression. Moreover, TME is implicated in malignant cell protection against anti-tumor therapy. This review describes the main cellular and non-cellular components located in the BM, which condition the immunosuppressive environment and lead the MM establishment and progression.

Background:The 5-year survival rate of patients with pancreatic ductal adenocarcinoma (PDAC) is around 5% due to the fact that the majority of patients present with advanced disease that is treatment resistant. Familial pancreatic cancer (FPC) is a rare disorder that is defined as a family with at least two affected first degree relatives, with an estimated incidence of 4%À10%. The genetic basis is unknown in the majority of families although around 10%À13% of families carry germline mutations in known genes associated with hereditary cancer and pancreatitis syndromes. Methods: Panel sequencing was performed of 35 genes associated with hereditary cancer in 43 PDAC cases from families with an apparent hereditary pancreatic cancer syndrome. Findings: Pathogenic variants were identified in 19% (5/26) of PDAC cases from pure FPC families in the genes MLH1, CDKN2A, POLQ and FANCM. Low frequency potentially pathogenic VUS were also identified in 35% (9/26) of PDAC cases from FPC families in the genes FANCC, MLH1, PMS2, CFTR, APC and MUTYH. Furthermore, an important proportion of PDAC cases harboured more than one pathogenic, likely pathogenic or potentially pathogenic VUS, highlighting the multigene phenotype of FPC. Interpretation: The genetic basis of familial or hereditary pancreatic cancer can be explained in 21% of families by previously described hereditary cancer genes. Low frequency variants in other DNA repair genes are also present in 35% of families which may contribute to the risk of pancreatic cancer development. Funding: This study was funded by the Instituto de Salud Carlos III (Plan Estatal de I + D + i 2013À2016): ISCIII (PI09/02221, PI12/01635, PI15/02101 and PI18/1034) and co-financed by the European Development Regional Fund ''A way to achieve Europe'' (ERDF), the Biomedical Research Network in Cancer: CIBERONC (CB16/12/00446), Red Tem atica de investigaci on cooperativa en c ancer: RTICC (RD12/0036/0073) and La Asociaci on Española contra el C ancer: AECC (Grupos Coordinados Estables 2016).

We previously reported a new approach for micromanipulation and encapsulation of human stem cells using a droplet-based microfluidic device. This approach demonstrated the possibility of encapsulating and culturing difficult-to-preserve primary human hematopoietic stem cells using an engineered double-layered bead composed by an inner layer of alginate and an outer layer of Puramatrix. We also demonstrated the maintenance and expansion of Multiple Myeloma cells in this construction. Here, the presented microfluidic technique is applied to construct a 3D biomimetic model to recapitulate the human hematopoietic stem cell niche using double-layered hydrogel beads cultured in 10% FBS culture medium. In this model, the long-term maintenance of the number of cells and expansion of hHSCS encapsulated in the proposed structures was observed. Additionally, a phenotypic characterization of the human hematopoietic stem cells generated in the presented biomimetic model was performed in order to assess their long-term stemness maintenance. Results indicate that the ex vivo cultured human CD34+ cells from bone marrow were viable, maintained, and expanded over a time span of eight weeks. This novel long-term stem cell culture methodology could represent a novel breakthrough to improve Hematopoietic Progenitor cell Transplant (HPT) as well as a novel tool for further study of the biochemical and biophysical factors influencing stem cell behavior. This technology opens a myriad of new applications as a universal stem cell niche model potentially able to expand other types of cells.

Despite the impressive results of autologous CAR-T cell therapy in refractory B lymphoproliferative diseases, CAR-NK immunotherapy emerges as a safer, faster, and cost-effective approach with no signs of severe toxicities as described for CAR-T cells. Permanently scrutinized for its efficacy, recent promising data in CAR-NK clinical trials point out the achievement of deep, high-quality responses, thus confirming its potential clinical use. Although CAR-NK cell therapy is not significantly affected by the loss or downregulation of its CAR tumor target, as in the case of CAR-T cell, a plethora of common additional tumor intrinsic or extrinsic mechanisms that could also disable NK cell function have been described. Therefore, considering lessons learned from CAR-T cell therapy, the emergence of CAR-NK cell therapy resistance can also be envisioned. In this review we highlight the processes that could be involved in its development, focusing on cytokine addiction and potential fratricide during manufacturing, poor tumor trafficking, exhaustion within the tumor microenvironment (TME), and NK cell short in vivo persistence on account of the limited expansion, replicative senescence, and rejection by patient’s immune system after lymphodepletion recovery. Finally, we outline new actively explored alternatives to overcome these resistance mechanisms, with a special emphasis on CRISPR/Cas9 mediated genetic engineering approaches, a promising platform to optimize CAR-NK cell function to eradicate refractory cancers.

Background:Chimeric antigen receptors (CARs) have been used in the past several years in cancer therapy to redirect immune effector cells. Despite impressive preliminary efficacy of CAR‐T cells in multiple myeloma (MM), NK cell engineering has emerged as a competitive and safer approach. NK‐92 is a universal, cheap and fast “off‐the‐shelf” cellular therapeutic previously used in clinical trials. Although modest responses with these cells have been reported in MM, their oncolytic potential can be enhanced by genetic modification. So far, two preclinical studies have been performed with CAR NK‐92 against MM, targeting CD138 or CS1 (SLAMF7). However, there are still reasonable doubts about its clinical outcomes due to on‐target off‐tumor effect or fratricide, respectively.Aims:Thus, the aim of our study is to generate and compare two novel CAR NK‐92 products for MM treatment.Methods:NK‐92MI cells were lentivirally transduced with the full‐length ectodomain sequence of the human native NKG2D receptor or with an anti‐BCMA scFv, both containing identical 4–1BB costimulatory and CD3‐ζ signaling domains (Figure 1A). To compare the efficacy between these 2nd generation NKG2D‐CAR NK‐92MI and BCMA‐CAR NK‐92MI cells, the same MOI 10 was used to transduce them; both populations were then purified by FACS sorting to obtain stable modified cell lines and vector copy number was measured by qPCR to ensure similar CAR expression. Cytotoxicity assays were performed by 3‐hours Calcein‐AM analysis. We used MM cell lines with different expression of target ligands: U266, BCMAhigh and NKG2DLhigh; XG‐1, a NK resistant cell line, BCMAhigh and NKG2DLlow; NCI H929 R20, a bortezomib resistant cell line with NKG2DLlow. K562, a leukemia cell line with BCMAnegative and NKG2DLhigh expression, was used as negative control.Results:NKG2D‐CAR NK‐92MI cells consistently showed much higher in vitro antitumor activity than the parental line NK‐92MI against U266 (84 ± 2% vs 40.7 ± 4% at a 1:1 E:T ratio), XG‐1 (67.9 ± 9% vs 18.5 ± 4% at a 16:1 E:T ratio) and NCI H929 #R20 (50.9 ± 6% vs 23.7 ± 2% at a 16:1 E:T ratio) cell lines (Figure 1B). NKG2D‐CAR NK‐92MI proved specificity in a blocking assay with an anti‐NKG2D antibody (82% decrease compares to baseline lysis). Furthermore, the oncolytic potential of NKG2D‐CAR NK‐92MI was not altered by physiological levels of soluble MICA, described in MM patients, or mandatory 10Gy irradiation prior to clinical use. Next, we compared cytotoxicity between NKG2D‐CAR NK‐92MI and BCMA‐CAR NK‐92MI against U‐266 (84 ± 2% vs 91.9 ± 3% at a 1:1 E:T ratio), XG‐1 (67.9 ± 9% vs 89.9 ± 2% at a 16:1 E:T ratio) and K562 (94 ± 3% vs 25.74 ± 4% at a 1:1 E:T ratio) cell lines (Figure 1C). Strikingly, there were no significant differences between NKG2D‐CAR NK and the gold standard BCMA‐CAR against MM cell lines with high and similar BCMA and NKG2DL expression. In addition, correlation between target ligands expression on the tumor and efficacy and specificity of both CARs was also shown. None of the CAR NK‐92MI studied populations showed toxicity against PBMCs from healthy donors and in vivo MM orthotopic xenograft mouse model experiments are ongoing.Summary/Conclusion:We have generated two novel and stable CAR NK‐92 immunoproducts that improve the oncolytic efficacy of the parental cell line. Indeed, NKG2D‐CAR NK‐92MI cells are as equally efficient as BCMA‐CAR NK‐92MI cells to eradicate diverse MM cells. To summarize, all these data show the feasibility to use this NK ‘off‐the‐shelf’ approach as immunotherapy for MM.image

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Multiple myeloma (MM) is characterized by an immunosuppressive microenvironment that enables tumor development. One of the mechanisms of immune evasion used by MM cells is the inhibition of natural killer (NK) cell effector functions; thus, the restoration of NK cell antitumor activity represents a key goal for new immunotherapeutic approaches, increasing tumor cell recognition, avoiding tumor escape and potentially enhancing the effect of other drugs. In this study, we evaluated the ability of the investigational medicine NKTR-255, an IL-15 receptor agonist, to engage the IL-15 pathway and stimulate NK cells against MM cells. We observed that incubation with NKTR-255 was able to tilt the balance towards an activated phenotype in NK cells isolated from peripheral blood mononuclear cells of MM patients, with increased expression of activating receptors on the surface of treated NK cells. This resulted in an enhanced degranulation, cytokine release and anti-tumor cytotoxicity when the NK cells were exposed to both MM cell lines and primary MM cells. We further evaluated the in vivo effect of NKTR-255 in fully humanized immunocompetent mice subcutaneously engrafted with H929 MM cells. Compared to placebo, weekly injection of the mice with NKTR-255 increased the number of circulating NK cells in peripheral blood and delayed tumor growth. Finally, we observed that combination of NKTR-255 with the anti-CD38 antibody, daratumumab, was effective against MM cells in vitro and in vivo. Taken together, our data suggest a significant impact of NKTR-255 in inducing NK cell function against MM cells with important translational implications.

Renal cells need oxygen for homeostasis; it is known for adjusting cellular functioning and the energy obtainment have a broad relationship with cellular respiration, through the O2 bioavailability. O2 homeostasis regulation in the kidney is mediated by hypoxia‐inducible factors (HIFs). HIF is divided into three α isoforms, represented by HIF‐1α, HIF‐2α, and HIF‐3α in addition to three paralogs of HIF‐1β; these are involved in some metabolic processes, as well as in the pathogenesis of several diseases. Renal biopsy analyses of patients and experimental animal models aim to understand the relationship between HIF and protection against developing renal diseases or the induction of their onset, being thus this molecule can be considered a potential biomarker of renal disease. We carried out a systematic review to which we included studies on HIF‐1α and renal disease in the last 5 years (2013‐2018) in researches with humans and/or animal model through searches in three databases: LILACS, PubMed, and SciELO by two researchers. We obtained 22 articles that discussed the relationship with HIF as inductor or protector against renal disease and no relation between HIF and renal. We observed controversies remain regarding the relation between of HIF with renal diseases; this may be related to the different intracellular pathways mediated by HIF‐1α, thereby determining differentiated cellular responses.