IL-27-engineered CAR.19-NK-92 cells exhibit enhanced therapeutic efficacy.

Chimeric antigen receptor (CAR) engineering of natural killer (NK) cells has shown promising results in early-phase clinical studies. However, advancing CAR-NK cell therapeutic efficacy is imperative. In this study, we investigated the impact of a fourth-generation CD19-targeted CAR (CAR.19) coexpressing IL-27 on NK-92 cells. We observed a significant improvement in NK-92 cell proliferation and cytotoxicity activity against B-cell cancer cell lines, both in vitro and in a xenograft mouse B-cell lymphoma model. Our systematic transcriptome analysis of the activated NK-92 CAR variants further supports the potential of IL-27 in fourth-generation CARs to overcome limitations of NK cell-based targeted tumor therapies by providing essential growth and activation signals. Integrating IL-27 into CAR-NK cells emerges as a promising strategy to enhance their therapeutic potential and elicit robust responses against cancer cells. These findings contribute substantially to the mounting evidence supporting the potential of fourth-generation CAR engineering in advancing NK cell-based immunotherapies.

PARP1 Characterization as a Potential Biomarker for BCR::ABL1 p190+ Acute Lymphoblastic Leukemia.

Detection of t(9;22), and consequent BCR::ABL1 fusion, is still a marker of worse prognosis for acute lymphoblastic leukemia (ALL), with resistance to tyrosine-kinase inhibitor therapy being a major obstacle in the clinical practice for this subset of patients. In this study, we investigated the effectiveness of targeting poly-ADP-ribose polymerase (PARP) in a model of BCR::ABL1 p190+ ALL, the most common isoform to afflict ALL patients, and demonstrated the use of experimental PARP inhibitor (PARPi), AZD2461, as a therapeutic option with cytotoxic capabilities similar to that of imatinib, the current gold standard in medical care. We characterized cytostatic profiles, induced cell death, and biomarker expression modulation utilizing cell models, also providing a comprehensive genome-wide analysis through an aCGH of the model used, and further validated PARP1 differential expression in samples of ALL p190+ patients from local healthcare institutions, as well as in larger cohorts of online and readily available datasets. Overall, we demonstrate the effectiveness of PARPi in the treatment of BCR::ABL1 p190+ ALL cell models and that PARP1 is differentially expressed in patient samples. We hope our findings help expand the characterization of molecular profiles in ALL settings and guide future investigations into novel biomarker detection and pharmacological choices in clinical practice.

Engineering NK-CAR.19 cells with the IL-15/IL-15Rα complex improved proliferation and anti-tumor effect in vivo.

Introduction: Natural killer 92 (NK-92) cells are an attractive therapeutic approach as alternative chimeric antigen receptor (CAR) carriers, different from T cells, once they can be used in the allogeneic setting. The modest in vivo outcomes observed with NK-92 cells continue to present hurdles in successfully translating NK-92 cell therapies into clinical applications. Adoptive transfer of CAR-NK-92 cells holds out the promise of therapeutic benefit at a lower rate of adverse events due to the absence of GvHD and cytokine release syndrome. However, it has not achieved breakthrough clinical results yet, and further improvement of CAR-NK-92 cells is necessary. Methods: In this study, we conducted a comparative analysis between CD19-targeted CAR (CAR.19) co-expressing IL-15 (CAR.19-IL15) with IL-15/IL-15Rα (CAR.19-IL15/IL15Rα) to promote NK cell proliferation, activation, and cytotoxic activity against B-cell leukemia. CAR constructs were cloned into lentiviral vector and transduced into NK-92 cell line. Potency of CAR-NK cells were assessed against CD19-expressing cell lines NALM-6 or Raji in vitro and in vivo in a murine model. Tumor burden was measured by bioluminescence. Results: We demonstrated that a fourth- generation CD19-targeted CAR (CAR.19) co-expressing IL-15 linked to its receptor IL-15/IL-15Rα (CAR.19-IL-15/IL-15Rα) significantly enhanced NK-92 cell proliferation, proinflammatory cytokine secretion, and cytotoxic activity against B-cell cancer cell lines in vitro and in a xenograft mouse model. Conclusion: Together with the results of the systematic analysis of the transcriptome of activated NK-92 CAR variants, this supports the notion that IL-15/IL-15Rα comprising fourth-generation CARs may overcome the limitations of NK-92 cell-based targeted tumor therapies in vivo by providing the necessary growth and activation signals.

Genome Editing for Engineering the Next Generation of Advanced Immune Cell Therapies.

Our current genetic engineering capacity through synthetic biology and genome editing is the foundation of a revolution in biomedical science: the use of genetically programmed cells as therapeutics. The prime example of this paradigm is the adoptive transfer of genetically engineered T cells to express tumor-specific receptors, such as chimeric antigen receptors (CARs) or engineered T-cell receptors (TCR). This approach has led to unprecedented complete remission rates in patients with otherwise incurable hematological malignancies. However, this approach is still largely ineffective against solid tumors, which comprise the vast majority of neoplasms. Also, limitations associated with the autologous nature of this therapy and shared markers between cancer cells and T cells further restrict the access to these therapies. Here, we described how cutting-edge genome editing approaches have been applied to unlock the full potential of these revolutionary therapies, thereby increasing therapeutic efficacy and patient accessibility.

BCR-ABL promotes hematopoietic stem and progenitor cell formation in embryonic stem cells.

Generating hematopoietic stem cells (HSCs) from pluripotent stem cells (PSCs) has been a long-lasting quest in the field of hematopoiesis. Previous studies suggested that enforced expression of BCR-ABL, the unique oncogenic driver of chronic myelogeneous leukemia (CML), in embryonic stem cells (ESCs)-derived hematopoietic cells is sufficient to confer long-term in vivo repopulating potential. To precisely uncover the molecular events regulated by the tyrosine kinase activity of BCR-ABL1 (p210) during the course of hematopoietic differentiation, we engineered a Tet-ON inducible system to modulate its expression in murine ESCs (mESCs). We showed in unique site-directed knock-in ESC model that BCR-ABL expression tightly regulated by doxycycline (dox) controls the formation and the maintenance of immature hematopoietic progenitors. Interestingly, these progenitors can be expanded in vitro for several passages in the presence of dox. Our analysis of cell surface markers and transcriptome compared with wild-type fetal and adult HSCs unraveled a similar molecular signature. Long-term culture initiating cell (LTC-IC) assay confirmed their self-renewal capacities albeit with a differentiation bias toward erythroid and myeloid cells. Collectively, our novel Tet-ON system represents a unique in vitro model to shed lights on ESC-derived hematopoiesis, CML initiation, and maintenance.

Association between Immunophenotypic Parameters and Molecular Alterations in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is a hematologic malignancy that occurs due to alterations such as genetic mutations, chromosomal translocations, or changes in molecular levels. These alterations can accumulate in stem cells and hematopoietic progenitors, leading to the development of AML, which has a prevalence of 80% of acute leukemias in the adult population. Recurrent cytogenetic abnormalities, in addition to mediating leukemogenesis onset, participate in its evolution and can be used as established diagnostic and prognostic markers. Most of these mutations confer resistance to the traditionally used treatments and, therefore, the aberrant protein products are also considered therapeutic targets. The surface antigens of a cell are characterized through immunophenotyping, which has the ability to identify and differentiate the degrees of maturation and the lineage of the target cell, whether benign or malignant. With this, we seek to establish a relationship according to the molecular aberrations and immunophenotypic alterations that cells with AML present.

Hybrid lipid-biopolymer nanocarrier as a strategy for GBM photodynamic therapy (PDT).

Glioblastoma (GBM) is the most common brain cancer characterized by aggressive and infiltrated tumors. For this, hybrid biopolymer-lipid nanoparticles coated with biopolymers such as chitosan and lipidic nanocarriers (LN) loaded with a photosensitizer (AlClPc) can be used for GBM photodynamic therapy. The chitosan-coated LN exhibited stable physicochemical characteristics and presented as an excellent lipid nanocarrier with highly efficiently encapsulated photosensitizer chloro-aluminum phthalocyanine (AlClPc). LN(AlClPc)Ct0.1% in the presence of light produced more reactive oxygen species and reduced brain tumor cell viability and proliferation. Confirm the effects of in vivo LN applications with photodynamic therapy confirmed that the total brain tumor area decreased without systemic toxicity in mice. These results suggest a promising strategy for future clinical applications to improve brain cancer treatment.

miR-495-3p sensitizes BCR-ABL1-expressing leukemic cells to tyrosine kinase inhibitors by targeting multidrug resistance 1 gene in T315I mutated cells.

Chronic myeloid leukemia (CML) is a clonal hematopoietic malignancy driven by the BCR-ABL1 fusion oncoprotein. The development of tyrosine kinase inhibitors (TKIs) has deeply increased long-term survival of CML patients. Nonetheless, one patient out of four will switch TKI off owing either to drug intolerance or resistance partly due to amplification or mutations of BCR-ABL1 oncogene and alteration in ATP-binding cassette (ABC) transporters. Increasing evidence suggests the involvement of the microRNA miR-495-3p in cancer-associated chemoresistance through multidrug resistance 1 (MDR1) gene, which encodes an ATP-dependent efflux pump. Our study aimed at investigating the potential role of miR-495-3p in CML TKI chemo-sensitivity and determining the underlying molecular circuitry involved. We first observed that miR-495-3p expression was lower in BCR-ABL1-expressing cellular models in vitro. Notably, loss-of-function experiments showed increased proliferation associated with a decreased number of nondividing cells (G0/G1) and resistance to Imatinib. Conversely, our data showed that miR-495-3p overexpression hindered leukemic cell growth and TKI resistance in Imatinib-resistant T315I-mutant cells, as well as drug efflux activity through MDR1 regulation. Further investigating the role of miR-495-3p in CML patients, we found that predicted miR-495-3p targets were upregulated in patients in blast crisis that were involved in protein phosphorylation and associated with the worst prognosis. Taken together, our results demonstrate that downregulation of miR-495-3p expression is important in the malignant phenotype of CML and TKI resistance mechanisms and could be a useful biomarker and a potential therapeutic target to eradicate CML.

Chimeric antigen-receptor (CAR) engineered natural killer cells in a chronic myeloid leukemia (CML) blast crisis model.

During the last two decades, the introduction of tyrosine kinase inhibitors (TKIs) to the therapy has changed the natural history of CML but progression into accelerated and blast phase (AP/BP) occurs in 3-5% of cases, especially in patients resistant to several lines of TKIs. In TKI-refractory patients in advanced phases, the only curative option is hematopoietic stem cell transplantation. We and others have shown the relevance of the expression of the Interleukin-2-Receptor α subunit (IL2RA/CD25) as a biomarker of CML progression, suggesting its potential use as a therapeutic target for CAR-based therapies. Here we show the development of a CAR-NK therapy model able to target efficiently a blast crisis cell line (K562). The design of the CAR was based on the scFv of the clinically approved anti-CD25 monoclonal antibody (Basiliximab). The CAR construct was integrated into NK92 cells resulting in the generation of CD25 CAR-NK92 cells. Target K562 cells were engineered by lentiviral gene transfer of CD25. In vitro functionality experiments and in vivo leukemogenicity experiments in NSG mice transplanted by K562-CD25 cells showed the efficacy and specificity of this strategy. These proof-of-concept studies could represent a first step for further development of this technology in refractory/relapsed (R/R) CML patients in BP as well as in R/R acute myeloblastic leukemias (AML).

Sex and Circadian Timing Modulate Oxaliplatin Hematological and Hematopoietic Toxicities.

Oxaliplatin was nearly twice as hematotoxic, with optimal circadian timing differing by 6 h, in women as compared to men with colorectal cancers. Hence, we investigated sex- and timing-related determinants of oxaliplatin hematopoietic toxicities in mice. Body-weight loss (BWL), blood cell counts, bone marrow cellularity (BMC) and seven flow-cytometry-monitored hematopoietic progenitor populations were evaluated 72 h after oxaliplatin chronotherapy administration (5 mg/kg). In control animals, circadian rhythms of circulating white blood cells showed a peak at ZT5 in both sexes, whereas BMC was maximum at ZT20 in males and ZT13h40 in females. All BM progenitor counts presented robust rhythms with phases around ZT3h30 in females, whereas only three of them rhythmically cycled in males with a ≈ -6 h phase shift. In treated females, chronotoxicity rhythms occurred in BWL, WBC, BMC and all BM progenitors with the best timing at ZT15, ZT21, ZT15h15 and ZT14h45, respectively. In males, almost no endpoints showed circadian rhythms, BWL and WBC toxicity being minimal, albeit with a substantial drop in BM progenitors. Increasing dose (10 mg/kg) in males induced circadian rhythms in BWL and WBC but not in BM endpoints. Our results suggest complex and sex-specific clock-controlled regulation of the hematopoietic system and its response to oxaliplatin.

Combination of genetically engineered T cells and immune checkpoint blockade for the treatment of cancer.

Immune checkpoint (IC) blockade using monoclonal antibodies is currently one of the most successful immunotherapeutic interventions to treat cancer. By reinvigorating antitumor exhausted T cells, this approach can lead to durable clinical responses. However, the majority of patients either do not respond or present a short-lived response to IC blockade, in part due to a scarcity of tumor-specific T cells within the tumor microenvironment. Adoptive transfer of T cells genetically engineered to express chimeric antigen receptors (CARs) or engineered T-cell receptors (TCRs) provide the necessary tumor-specific immune cell population to target cancer cells. However, this therapy has been considerably ineffective against solid tumors in part due to IC-mediated immunosuppressive effects within the tumor microenvironment. These limitations could be overcome by associating adoptive cell transfer of genetically engineered T cells and IC blockade. In this comprehensive review, we highlight the strategies and outcomes of preclinical and clinical attempts to disrupt IC signaling in adoptive T-cell transfer against cancer. These strategies include combined administration of genetically engineered T cells and IC inhibitors, engineered T cells with intrinsic modifications to disrupt IC signaling, and the design of CARs against IC molecules. The current landscape indicates that the synergy of the fast-paced refinements of gene-editing technologies and synthetic biology and the increased comprehension of IC signaling will certainly translate into a novel and more effective immunotherapeutic approaches to treat patients with cancer.

Leukemic Stem Cell: A Mini-Review on Clinical Perspectives.

Hematopoietic stem cells (HSCs) are known for their ability to proliferate and self-renew, thus being responsible for sustaining the hematopoietic system and residing in the bone marrow (BM). Leukemic stem cells (LSCs) are recognized by their stemness features such as drug resistance, self-renewal, and undifferentiated state. LSCs are also present in BM, being found in only 0.1%, approximately. This makes their identification and even their differentiation difficult since, despite the mutations, they are cells that still have many similarities with HSCs. Although the common characteristics, LSCs are heterogeneous cells and have different phenotypic characteristics, genetic mutations, and metabolic alterations. This whole set of alterations enables the cell to initiate the process of carcinogenesis, in addition to conferring drug resistance and providing relapses. The study of LSCs has been evolving and its application can help patients, where through its count as a biomarker, it can indicate a prognostic factor and reveal treatment results. The selection of a target to LSC therapy is fundamental. Ideally, the target chosen should be highly expressed by LSCs, highly selective, absence of expression on other cells, in particular HSC, and preferentially expressed by high numbers of patients. In view of the large number of similarities between LSCs and HSCs, it is not surprising that current treatment approaches are limited. In this mini review we seek to describe the immunophenotypic characteristics and mechanisms of resistance presented by LSCs, also approaching possible alternatives for the treatment of patients.

Circadian Rhythm Dysregulation and Leukemia Development: The Role of Clock Genes as Promising Biomarkers.

The circadian clock (CC) is a daily system that regulates the oscillations of physiological processes and can respond to the external environment in order to maintain internal homeostasis. For the functioning of the CC, the clock genes (CG) act in different metabolic pathways through the clock-controlled genes (CCG), providing cellular regulation. The CC's interruption can result in the development of different diseases, such as neurodegenerative and metabolic disorders, as well as cancer. Leukemias correspond to a group of malignancies of the blood and bone marrow that occur when alterations in normal cellular regulatory processes cause the uncontrolled proliferation of hematopoietic stem cells. This review aimed to associate a deregulated CC with the manifestation of leukemia, looking for possible pathways involving CG and their possible role as leukemic biomarkers.

BMAL1 Knockdown Leans Epithelial-Mesenchymal Balance toward Epithelial Properties and Decreases the Chemoresistance of Colon Carcinoma Cells.

The circadian clock coordinates biological and physiological functions to day/night cycles. The perturbation of the circadian clock increases cancer risk and affects cancer progression. Here, we studied how BMAL1 knockdown (BMAL1-KD) by shRNA affects the epithelial-mesenchymal transition (EMT), a critical early event in the invasion and metastasis of colorectal carcinoma (CRC). In corresponding to a gene set enrichment analysis, which showed a significant enrichment of EMT and invasive signatures in BMAL1_high CRC patients as compared to BMAL1_low CRC patients, our results revealed that BMAL1 is implicated in keeping the epithelial-mesenchymal equilibrium of CRC cells and influences their capacity of adhesion, migration, invasion, and chemoresistance. Firstly, BMAL1-KD increased the expression of epithelial markers (E-cadherin, CK-20, and EpCAM) but decreased the expression of Twist and mesenchymal markers (N-cadherin and vimentin) in CRC cell lines. Finally, the molecular alterations after BMAL1-KD promoted mesenchymal-to-epithelial transition-like changes mostly appeared in two primary CRC cell lines (i.e., HCT116 and SW480) compared to the metastatic cell line SW620. As a consequence, migration/invasion and drug resistance capacities decreased in HCT116 and SW480 BMAL1-KD cells. Together, BMAL1-KD alerts the delicate equilibrium between epithelial and mesenchymal properties of CRC cell lines, which revealed the crucial role of BMAL1 in EMT-related CRC metastasis and chemoresistance.

Strategies to Enhance the Therapeutic Efficacy, Applicability, and Safety of Genetically Engineered Immune Cells.

The field of cell therapy is leading a paradigm shift in drug development. The recent convergence of several fields, including immunology, genetics, and synthetic biology, now allows for the introduction of artificial receptors and the design of entire genetic circuitries to finely program the behavior of injected cells. A prime example of these next-generation living drugs comes in the form of T cells expressing chimeric antigen receptors (CARs), which have already demonstrated definitive evidence of therapeutic efficacy against some hematological malignancies. However, several obstacles still restrict the antitumor efficacy of and impair the widespread use of CAR-T cells. Critical challenges include limited persistence and antitumor activity in vivo, antigen escape, scarcity of suitable single markers for targeting, and therapy-related toxicity. Nevertheless, intense research activity in this field has resulted in a plethora of creative solutions to address each of these limitations. In this review, we provide a comprehensive snapshot of the current strategies used to enhance the therapeutic efficacy, applicability, and safety of genetically engineered immune cells to treat cancer.

Human and mouse melanoma cells recapitulate an EMT-like program in response to mesenchymal stromal cells secretome.

The mechanisms underlying the propensity of melanomas to metastasize are not completely understood. We hypothesized that melanoma cells are capable of promptly activating an epithelial-to-mesenchymal transition (EMT)-like profile in response to stroma-derived factors. Thus, we investigated the role of mesenchymal stromal cells (MSCs), a cell population considered as a precursor of tumor stroma, on the activation of an EMT-like profile and acquisition of metastatic traits in melanoma cells. After subcutaneous co-injection with mouse B16 melanoma cells, MSCs occupied perivascular sites within tumors and enhanced B16 metastasis to the lungs. In vitro, MSCs' secretome activated an EMT-like profile in B16 cells, reducing their avidity to fibronectin, and increasing their motility and invasiveness. These effects were abrogated upon blocking of MET phosphorylation in B16 cells using small molecule inhibitors. MSCs also activated an EMT-like profile in human melanoma cells from different stages of progression. Activation of EMT in human cells was associated with increased levels of p-STAT1 and p-STAT3. In conclusion, both mouse and human melanoma cells are equipped to activate an EMT-like program and acquire metastatic traits through the activation of distinct pathways by MSCs' secretome.

NTAL is associated with treatment outcome, cell proliferation and differentiation in acute promyelocytic leukemia.

Non-T cell activation linker (NTAL) is a lipid raft-membrane protein expressed by normal and leukemic cells and involved in cell signaling. In acute promyelocytic leukemia (APL), NTAL depletion from lipid rafts decreases cell viability through regulation of the Akt/PI3K pathway. The role of NTAL in APL cell processes, and its association with clinical outcome, has not, however, been established. Here, we show that reduced levels of NTAL were associated with increased all-trans retinoic acid (ATRA)-induced differentiation, generation of reactive oxygen species, and mitochondrial dysfunction. Additionally, NTAL-knockdown (NTAL-KD) in APL cell lines led to activation of Ras, inhibition of Akt/mTOR pathways, and increased expression of autophagy markers, leading to an increased apoptosis rate following arsenic trioxide treatment. Furthermore, NTAL-KD in NB4 cells decreased the tumor burden in (NOD scid gamma) NSG mice, suggesting its implication in tumor growth. A retrospective analysis of NTAL expression in a cohort of patients treated with ATRA and anthracyclines, revealed that NTAL overexpression was associated with a high leukocyte count (P = 0.007) and was independently associated with shorter overall survival (Hazard Ratio: 3.6; 95% Confidence Interval: 1.17-11.28; P = 0.026). Taken together, our data highlights the importance of NTAL in APL cell survival and response to treatment.

Image and motor behavior for monitoring tumor growth in C6 glioma model.

The primary objective of this study is to monitor tumor growth by using image techniques and behavioral testing through general and specific motor activities (spontaneous movements and gait). Our sample includes male Wistar rats, 2 months old and weighing 250-300 g, that is categorized into three groups: control, sham, and experimental. The experimental group was anesthetized; the C6 cells with luciferase expression that were suspended in a culture medium were implanted into the right frontoparietal cortex of the rats. The sham group received implant only with culture medium without cells. Images and behavioral tests were evaluated at base time and at 7, 14, 21, and 28 days after induced tumor growth analysis. The tumor volume measured by magnetic resonance imaging (MRI) and quantitative bioluminescence imaging (BLI) signal showed a correlation coefficient of r = 0.96. The MRI showed that the mean tumor volume increased by approximately 10, 26, and 49 times according to a comparison of tumor volume on the seventh day with 14, 21, and 28 days, respectively. The quantification of the BLI signal was (4.12 ± 2.01) x 10(8), (8.33 ± 3.12) x 10(8), (28.43 ± 6.32) x 10(8), and (63.02 ± 10.53) x 10(8) photons/s at the seventh, fourteenth, twenty-first, and twenty-eighth day, respectively. After 14 days of tumor induction, both behavioral tests showed significant differences between tumor and sham or control groups. Our study showed a high correlation between MRI and BLI for tumor growth monitoring with complement aspects analysis in tumor volume. In addition, functional behavioral analysis displayed sensitivity to monitor tumor growth, as well as to detect early significant changes between groups, primarily in the tumor group. The results of gait analysis were more sensitive than general motor analysis.

Mesenchymal Stem Cells and Pericytes: To What Extent Are They Related?

Mesenchymal stem cells (MSCs) were initially identified as progenitors of skeletal tissues within mammalian bone marrow and cells with similar properties were also obtained from other tissues such as adipose and dental pulp. Although MSCs have been extensively investigated, their native behavior and in vivo identity remain poorly defined. Uncovering the in vivo identity of MSCs has been challenging due to the lack of exclusive cell markers, cellular alterations caused by culture methods, and extensive focus on in vitro properties for characterization. Although MSC site of origin influences their functional properties, these mesenchymal progenitors can be found in the perivascular space in virtually all organs from where they were obtained. However, the precise identity of MSCs within the vascular wall is highly controversial. The recurrent concept that MSCs correspond to pericytes in vivo has been supported mainly by their perivascular localization and expression of some molecular markers. However, this view has been a subject of controversy, in part, due to the application of loose criteria to define pericytes and due to the lack of a marker able to unequivocally identify these cells. Furthermore, recent evidences indicate that subpopulations of MSCs can be found at extravascular sites such as the endosteum. In this opinion review, we bring together the advances and pitfalls on the search for the in vivo identity of MSCs and highlight the recent evidences that suggest that perivascular MSCs are adventitial cells, acting as precursors of pericytes and other stromal cells during tissue homeostasis.

Therapeutic efficacy and biodistribution of allogeneic mesenchymal stem cells delivered by intrasplenic and intrapancreatic routes in streptozotocin-induced diabetic mice.

INTRODUCTION: Mesenchymal stromal/stem cells (MSCs) are multipotent cells that have the ability to express and secrete a wide range of immunomodulatory molecules, cytokines, growth factors and antiapoptotic proteins. MSCs modulate both innate and adaptive immune responses making them potential candidates for the treatment of patients with type 1 diabetes mellitus (T1D). However, one problem frequently associated with the systemic MSCs administration is the entrapment of the cells mainly in the lungs. In this sense, trying to avoid the lung barrier, the purpose of this study was to evaluate the long-term therapeutic efficacy and biodistribution of allogeneic adipose tissue-derived MSCs (ADMSCs) injected via two different delivery routes (intrasplenic/I.Sp and intrapancreatic/I.Pc) in a murine model of diabetes induced by streptozotocin (STZ). METHODS: Experimental diabetes was induced in C57BL/6 male mice by multiple low-doses of STZ. MSCs were isolated from adipose tissue (ADMSCs) of Balb/c mice. A single dose of 1x10(6) ADMSCs was microinjected into the spleen or into the pancreas of diabetic mice. Control group received injection of PBS by I.Sp or I.Pc delivery routes. Glycemia, peripheral glucose response, insulin-producing ß cell mass, regulatory T cell population, cytokine profile and cell biodistribution were evaluated after ADMSCs/PBS administration. RESULTS: ADMSCs injected by both delivery routes were able to decrease blood glucose levels and improve glucose tolerance in diabetic mice. ADMSCs injected by I.Sp route reverted hyperglycemia in 70% of diabetic treated mice, stimulating insulin production by pancreatic ß cells. Using the I.Pc delivery route, 42% of ADMSCs-treated mice responded to the therapy. Regulatory T cell population remained unchanged after ADMSCs administration but pancreatic TGF-ß levels were increased in ADMSCs/I.Sp-treated mice. ADMSCs administrated by I.Sp route were retained in the spleen and in the liver and ADMSCs injected by I.Pc route remained in the pancreas. However, ADMSCs injected by these delivery routes remained only few days in the recipients. CONCLUSION: Considering the potential role of MSCs in the treatment of several disorders, this study reports alternative delivery routes that circumvent cell entrapment into the lungs promoting beneficial therapeutic responses in ADMSCs-treated diabetic mice.