Placental circulating T cells: a novel, allogeneic CAR-T cell platform with preserved T-cell stemness, more favorable cytokine profile, and durable efficacy compared to adult PBMC-derived CAR-T.

BACKGROUND: Chimeric antigen receptor (CAR)-T cell quality and stemness are associated with responsiveness, durability, and memory formation, which benefit clinical responses. Autologous T cell starting material across patients with cancer is variable and CAR-T expansion or potency can fail during manufacture. Thus, strategies to develop allogeneic CAR-T platforms including the identification and expansion of T cell subpopulations that correspond with CAR-T potency are an active area of investigation. Here, we compared CAR-T cells generated from healthy adult peripheral blood T cells versus placental circulating T (P-T) cells. METHODS: CAR-T cells from healthy adult peripheral blood mononuclear cells (PBMCs) and P-T cells were generated using the same protocol. CAR-T cells were characterized in detail by a combination of multiparameter flow cytometry, functional assays, and RNA sequencing. In vivo antitumor efficacy and persistence of CAR-T cells were evaluated in a Daudi lymphoma xenograft model. RESULTS: P-T cells possess stemness advantages compared with T cells from adult PBMCs. P-T cells are uniformly naïve prior to culture initiation, maintain longer telomeres, resist immune checkpoint upregulation, and resist further differentiation compared with PBMC T cells during CD19 CAR-T manufacture. P-T CD19 CAR-T cells are equally cytotoxic as PBMC-CD19 CAR-T cells but produce less interferon gamma in response to lymphoma. Transcriptome analysis shows P-T CD19 CAR-T cells retain a stem-like gene signature, strongly associate with naïve T cells, an early memory phenotype, and a unique CD4 T cell signature compared with PBMC-CD19 CAR-T cells, which enrich for exhaustion and stimulated memory T cell signatures. Consistent with functional data, P-T CD19 CAR-T cells exhibit attenuated inflammatory cytokine and chemokine gene signatures. In a murine in vivo model, P-T CD19 CAR-T cells eliminate lymphoma beyond 90 days. PBMC-CD19 CAR-T cells provide a non-durable benefit, which only delays disease onset. CONCLUSION: We identified characteristics of T cell stemness enriched in P-T CD19 CAR-T which are deficient in PBMC-derived products and translate into response durability in vivo. Our findings demonstrate that placental circulating T cells are a valuable cell source for allogeneic CAR-T products. Stemness advantages inherent to P-T cells translate to in vivo persistence advantages and long-term durable activity.

An in vitro comparison of human corneal epithelial cell activity and inflammatory response on differently designed ocular amniotic membranes and a clinical case study.

Amniotic membrane (AM) is a naturally derived biomaterial with biological and mechanical properties important to Ophthalmology. The epithelial side of the AM promotes epithelialization, while the stromal side regulates inflammation. However, not all AMs are equal. AMs undergo different processing with resultant changes in cellular content and structure. This study evaluates the effects of sidedness and processing on human corneal epithelial cell (HCEC) activity, the effect of processing on HCEC inflammatory response, and then a case study is presented. Three differently processed, commercially available ocular AMs were selected: (1) Biovance®3L Ocular, a decellularized, dehydrated human AM (DDHAM), (2) AMBIO2®, a dehydrated human AM (DHAM), and (3) AmnioGraft®, a cryopreserved human AM (CHAM). HCECs were seeded onto the AMs and incubated for 1, 4 and 7 days. Cell adhesion and viability were evaluated using alamarBlue assay. HCEC migration was evaluated using a scratch wound assay. An inflammatory response was induced by TNF-α treatment. The effect of AM on the expression of pro-inflammatory genes in HCECs was compared using quantitative polymerase chain reaction (qPCR). Staining confirmed complete decellularization and the absence of nuclei in DDHAM. HCEC activity was best supported on the stromal side of DDHAM. Under inflammatory stimulation, DDHAM promoted a higher initial inflammatory response with a declining trend across time. Clinically, DDHAM was used to successfully treat anterior basement membrane dystrophy. Compared with DHAM and CHAM, DDHAM had significant positive effects on the cellular activities of HCECs in vitro, which may suggest greater ocular cell compatibility in vivo.

Human placental hematopoietic stem cell-derived natural killer cells (CYNK) recognize and eliminate influenza A virus-infected cells.

Influenza A virus (IAV) infections are a significant recurrent threat to public health and a significant burden on global economy, highlighting the need for developing more effective therapies. Natural killer (NK) cells play a pivotal role in the control of pulmonary IAV infection, however, little is known about the therapeutic potential of adoptively transferred NK cells for viral infections. Here, we investigated the antiviral activity of CYNK, human placental hematopoietic stem cell-derived NK cells, against IAV infection in vitro. Virus infection induced the expression of NK cell activating ligands on respiratory epithelial cells, resulting in enhanced recognition by CYNK cells. Upon co-culture with IAV-infected epithelial cells, CYNK exhibited elevated degranulation and increased production of IFN-γ, TNF-α and GM-CSF in a virus dose-dependent manner. Furthermore, CYNK showed virus dose-dependent cytotoxicity against IAV-infected cells. The antiviral activity of CYNK was mediated by NKp46 and NKG2D. Together, these data demonstrate that CYNK possesses potent antiviral function against IAV and warrant clinical investigations for adoptive NK cell therapies against viral infections.

Human placental hematopoietic stem cell derived natural killer cells (CYNK-001) mediate protection against influenza a viral infection.

Influenza A virus (IAV) infections are associated with a high healthcare burden around the world and there is an urgent need to develop more effective therapies. Natural killer (NK) cells have been shown to play a pivotal role in reducing IAV-induced pulmonary infections in preclinical models; however, little is known about the therapeutic potential of adoptively transferred NK cells for IAV infections. Here, we investigated the effects of CYNK-001, human placental hematopoietic stem cell derived NK cells that exhibited strong cytolytic activity against a range of malignant cells and expressed high levels of activating receptors, against IAV infections. In a severe IAV-induced acute lung injury model, mice treated with CYNK-001 showed a milder body weight loss and clinical symptoms, which led to a delayed onset of mortality, thus demonstrating their antiviral protection in vivo. Analysis of bronchoalveolar lavage fluid (BALF) revealed that CYNK-001 reduced proinflammatory cytokines and chemokines highlighting CYNK-001's anti-inflammatory actions in viral induced-lung injury. Furthermore, CYNK-001-treated mice had altered immune responses to IAV with reduced number of neutrophils in BALF yet increased number of CD8+ T cells in the BALF and lung compared to vehicle-treated mice. Our results demonstrate that CYNK-001 displays protective functions against IAV via its anti-inflammatory and immunomodulating activities, which leads to alleviation of disease burden and progression in a severe IAV-infected mice model. The potential of adoptive NK therapy for IAV infections warrants clinical investigation.

CBLB ablation with CRISPR/Cas9 enhances cytotoxicity of human placental stem cell-derived NK cells for cancer immunotherapy.

BACKGROUND: Tumors often develop resistance to surveillance by endogenous immune cells, which include natural killer (NK) cells. Ex vivo activated and/or expanded NK cells demonstrate cytotoxicity against various tumor cells and are promising therapeutics for adoptive cancer immunotherapy. Genetic modification can further enhance NK effector cell activity or activation sensitization. Here, we evaluated the effect of the genetic deletion of ubiquitin ligase Casitas B-lineage lymphoma pro-oncogene-b (CBLB), a negative regulator of lymphocyte activity, on placental CD34+ cell-derived NK (PNK) cell cytotoxicity against tumor cells. METHODS: Using CRISPR/Cas9 technology, CBLB was knocked out in placenta-derived CD34+ hematopoietic stem cells, followed by differentiation into PNK cells. Cell expansion, phenotype and cytotoxicity against tumor cells were characterized in vitro. The antitumor efficacy of CBLB knockout (KO) PNK cells was tested in an acute myeloid leukemia (HL-60) tumor model in NOD-scid IL2R gammanull (NSG) mice. PNK cell persistence, biodistribution, proliferation, phenotype and antitumor activity were evaluated. RESULTS: 94% of CBLB KO efficacy was achieved using CRISPR/Cas9 gene editing technology. CBLB KO placental CD34+ cells differentiated into PNK cells with high cell yield and >90% purity determined by CD56+ CD3- cell identity. Ablation of CBLB did not impact cell proliferation, NK cell differentiation or phenotypical characteristics of PNK cells. When compared with the unmodified PNK control, CBLB KO PNK cells exhibited higher cytotoxicity against a range of liquid and solid tumor cell lines in vitro. On infusion into busulfan-conditioned NSG mice, CBLB KO PNK cells showed in vivo proliferation and maturation as evidenced by increased expression of CD16, killer Ig-like receptors and NKG2A over 3 weeks. Additionally, CBLB KO PNK cells showed greater antitumor activity in a disseminated HL60-luciferase mouse model compared with unmodified PNK cells. CONCLUSION: CBLB ablation increased PNK cell effector function and proliferative capacity compared with non-modified PNK cells. These data suggest that targeting CBLB may offer therapeutic advantages via enhancing antitumor activities of NK cell therapies.

Human Placenta-Derived Mesenchymal Stromal-Like Cells Enhance Angiogenesis via T Cell-Dependent Reprogramming of Macrophage Differentiation.

Peripheral arterial disease (PAD) is a leading cause of limb loss and mortality worldwide with limited treatment options. Mesenchymal stromal cell (MSC) therapy has demonstrated positive effects on angiogenesis in preclinical models and promising therapeutic efficacy signals in early stage clinical studies; however, the mechanisms underlying MSC-mediated angiogenesis remain largely undefined. Here, we investigated the mechanism of action of human placenta-derived MSC-like cells (PDA-002) in inducing angiogenesis using mice hind limb ischemia model. We showed that PDA-002 improved blood flow and promoted collateral vessel formation in the injured limb. Histological analysis demonstrated that PDA-002 increased M2-like macrophages in ischemic tissue. Analysis of the changes in functional T cell phenotype in the draining lymph nodes revealed that PDA-002 treatment was associated with the induction of cytokine and gene expression signatures of Th2 response. Angiogenic effect of PDA-002 was markedly reduced in Balb/c nude mice compared with wild type. This reduction in efficacy was reversed by T cell reconstitution, suggesting T cells are essential for PDA-002-mediated angiogenesis. Furthermore, effect of PDA-002 on macrophage differentiation was also T cell-dependent as a PDA-002-mediated M2-like macrophage skewing was only observed in wild type and T cell reconstituted nude mice, but not in nude mice. Finally, we showed that PDA-002-treated animals had enhanced angiogenic recovery in response to the second injury when PDA-002 no longer persisted in vivo. These results suggest that PDA-002 enhances angiogenesis through an immunomodulatory mechanism involving T cell-dependent reprogramming of macrophage differentiation toward M2-like phenotype. Stem Cells 2017;35:1603-1613.

Angiogenic properties of human placenta-derived adherent cells and efficacy in hindlimb ischemia.

OBJECTIVE: Human placenta-derived adherent cells (PDACs) are a culture-expanded, undifferentiated mesenchymal-like population from full-term placental tissue and were previously shown to possess anti-inflammatory and immunomodulatory properties. PDACs (formulated as PDA-002) are in clinical trials for peripheral arterial disease with diabetic foot ulcer. In the current study, we examined their angiogenic and tissue reparative properties. METHODS: The effects of PDACs on survival and tube formation of human umbilical vein endothelial cells (HUVECs) were tested using conditioned media and noncontact coculture. Angiogenic effects were assessed in the chick chorioallantoic membrane assay. Hindlimb ischemia (HLI) was induced in mice and rats by femoral artery transection, and blood flow and blood vessel density were monitored in vivo by laser Doppler and angiography in the ischemic and control limbs. Tissue damage and regeneration in HLI were examined in histologic sections of quadriceps muscle stained with hematoxylin and eosin, and newly synthesized blood vessels were detected by indoxyl-tetrazolium staining for alkaline phosphatase. RESULTS: PDACs enhanced the survival of serum-starved HUVECs and stimulated HUVEC tube formation, and in the chick chorioallantoic membrane assay, PDACs stimulated blood vessel formation. In HLI, intramuscular administration of PDACs resulted in improved blood flow and vascular density, and in quadriceps muscle, tissue regeneration and increased numbers of blood vessels were observed. CONCLUSIONS: PDACs exhibited various activities consistent with angiogenesis and tissue repair, supporting the continued investigation of this cell therapy as treatment for vascular disease-related indications.

Human Placenta-derived Cells (PDA-001) for the Treatment of Moderate-to-severe Crohn's Disease: A Phase 1b/2a Study.

BACKGROUND: PDA-001 (cenplacel-L), a preparation of placenta-derived mesenchymal-like adherent cells with immunomodulatory effects, previously demonstrated safety and tolerability in an open-label Crohn's disease (CD) study. The current phase 1b/2a study evaluated the safety and efficacy of PDA-001 in subjects with moderate-to-severe CD. METHODS: Subjects had active inflammation on colonoscopy or elevated fecal calprotectin and inadequate response to conventional therapy. Concomitant therapy with stable doses of immunomodulators and/or biologics was permitted. Subjects received 8 units of PDA-001 (1.5 × 10 cells per unit) in the phase 1b open-label study. In the phase 2a double-blind study, subjects were randomly assigned placebo, 1 unit, or 4 units of PDA-001 (2 infusions 1 wk apart). The primary endpoint was induction of clinical response (≥100 points and/or 25% decrease in Crohn's Disease Activity Index) at 4 and 6 weeks. RESULTS: Fifty subjects were enrolled (safety analysis, 50 subjects; efficacy analysis, 48 subjects). Four subjects received 8 units of PDA-001 (phase 1b study); 46 subjects were subsequently randomized to 1 or 4 units of PDA-001 or placebo (phase 2a study). The primary endpoint was achieved in 10/28 (36%) of PDA-001 subjects compared with placebo (0%, P = 0.026). Clinical remission was achieved in 4/28 (14%) of PDA-001 subjects compared with placebo (0%, P = 0.3). One treatment-related serious adverse event occurred (systemic hypersensitivity reaction at 8 units). In the phase 2a study, serious adverse events occurred in 9/28 (32%) of PDA-001 subjects and 1/16 (7%) of placebo subjects. CONCLUSIONS: A 2-infusion regimen of PDA-001 induced clinical response in subjects with moderate-to-severe CD. Additional studies are warranted.

Human placenta-derived adherent cells improve cardiac performance in mice with chronic heart failure.

Human placenta-derived adherent cells (PDACs) are a culture-expanded, undifferentiated mesenchymal-like population derived from full-term placental tissue, with immunomodulatory, anti-inflammatory, angiogenic, and neuroprotective properties. PDA-001 (cenplacel-L), an intravenous formulation of PDAC cells, is in clinical development for the treatment of autoimmune and inflammatory diseases. We tested the therapeutic effects of PDA-001 in mice with chronic heart failure (CHF). Three weeks after transaortic constriction surgery to induce CHF, the mice underwent direct intramyocardial (IM) or i.v. injection of PDA-001 at a high (0.5 × 10(6) cells per mouse), medium (0.5 × 10(5) cells per mouse), or low (0.5 × 10(4) cells per mouse) dose. The mice were sacrificed 4 weeks after treatment. Echocardiography and ventricular catheterization showed that IM injection of PDA-001 significantly improved left ventricular systolic and diastolic function compared with injection of vehicle or i.v. injection of PDA-001. IM injection of PDA-001 also decreased cardiac fibrosis, shown by trichrome staining in the vicinity of the injection sites. Low-dose treatment showed the best improvement in cardiac performance compared with the medium- and high-dose groups. In another independent study to determine the mechanism of action with bromodeoxyuridine labeling, the proliferation rates of endothelial cells and cardiomyocytes were significantly increased by low or medium IM dose PDA-001. However, no surviving PDA-001 cells were detected in the heart 1 month after injection. In vivo real-time imaging consistently revealed that the PDA-001 cells were detectable only within 2 days after IM injection of luciferase-expressing PDA-001. Together, these results have demonstrated the cardiac therapeutic potential of PDA-001, likely through a paracrine effect.

Human placenta-derived cells (PDA-001) for the treatment of adults with multiple sclerosis: a randomized, placebo-controlled, multiple-dose study.

BACKGROUND: Infusion of PDA-001, a preparation of mesenchymal-like cells derived from full-term human placenta, is a new approach in the treatment of patients with multiple sclerosis. OBJECTIVE: This safety study aimed to rule out the possibility of paradoxical exacerbation of disease activity by PDA-001 in patients with multiple sclerosis. METHODS: This was a phase 1b, multicenter, randomized, double-blind, placebo-controlled, 2-dose ranging study including patients with relapsing-remitting multiple sclerosis or secondary progressive multiple sclerosis. The study was conducted at 6 sites in the United States and 2 sites in Canada. Patients were randomized 3:1 to receive 2 low-dose infusions of PDA-001 (150×10(6) cells) or placebo, given 1 week apart. After completing this cohort, subsequent patients received high-dose PDA-001 (600×10(6) cells) or placebo. Monthly brain magnetic resonance imaging scans were performed. The primary end point was ruling out the possibility of paradoxical worsening of MS disease activity. This was monitored using Cutter׳s rule (≥5 new gadolinium lesions on 2 consecutive scans) by brain magnetic resonance imaging on a monthly basis for six months and also the frequency of multiple sclerosis relapse. RESULTS: Ten patients with relapsing-remitting multiple sclerosis and 6 with secondary progressive multiple sclerosis were randomly assigned to treatment: 6 to low-dose PDA-001, 6 to high-dose PDA-001, and 4 to placebo. No patient met Cutter׳s rule. One patient receiving high-dose PDA-001 had an increase in T2 and gadolinium lesions and in Expanded Disability Status Scale score during a multiple sclerosis flare 5 months after receiving PDA-001. No other patient had an increase in Expanded Disability Status Scale score>0.5, and most had stable or decreasing Expanded Disability Status Scale scores. With high-dose PDA-001, 1 patient experienced a grade 1 anaphylactoid reaction and 1 had grade 2 superficial thrombophlebitis. Other adverse events were mild to moderate and included headache, fatigue, infusion site reactions, and urinary tract infection. CONCLUSION: PDA-001 infusions were safe and well tolerated in relapsing-remitting multiple sclerosis and secondary progressive multiple sclerosis patients. No paradoxical worsening of lesion counts was noted with either dose.

Characterization and ex vivo Expansion of Human Placenta-Derived Natural Killer Cells for Cancer Immunotherapy.

Recent clinical studies suggest that adoptive transfer of donor-derived natural killer (NK) cells may improve clinical outcome in hematological malignancies and some solid tumors by direct anti-tumor effects as well as by reduction of graft versus host disease (GVHD). NK cells have also been shown to enhance transplant engraftment during allogeneic hematopoietic stem cell transplantation (HSCT) for hematological malignancies. The limited ex vivo expansion potential of NK cells from peripheral blood (PB) or umbilical cord blood (UCB) has however restricted their therapeutic potential. Here we define methods to efficiently generate NK cells from donor-matched, full-term human placenta perfusate (termed Human Placenta-Derived Stem Cell, HPDSC) and UCB. Following isolation from cryopreserved donor-matched HPDSC and UCB units, CD56+CD3- placenta-derived NK cells, termed pNK cells, were expanded in culture for up to 3 weeks to yield an average of 1.2 billion cells per donor that were >80% CD56+CD3-, comparable to doses previously utilized in clinical applications. Ex vivo-expanded pNK cells exhibited a marked increase in anti-tumor cytolytic activity coinciding with the significantly increased expression of NKG2D, NKp46, and NKp44 (p < 0.001, p < 0.001, and p < 0.05, respectively). Strong cytolytic activity was observed against a wide range of tumor cell lines in vitro. pNK cells display a distinct microRNA (miRNA) expression profile, immunophenotype, and greater anti-tumor capacity in vitro compared to PB NK cells used in recent clinical trials. With further development, pNK may represent a novel and effective cellular immunotherapy for patients with high clinical needs and few other therapeutic options.

Safety and tolerability of human placenta-derived cells (PDA001) in treatment-resistant crohn's disease: a phase 1 study.

BACKGROUND: The clinical utility of cellular therapies is being investigated in a broad range of therapeutic areas. This phase 1 study represents the first exploration of PDA001, a preparation of cells cultured from human placental tissue, in subjects with Crohn's disease. METHODS: Twelve subjects with active, moderate-to-severe Crohn's disease unresponsive to previous therapy were given 2 intravenous infusions of PDA001 1 week apart, monitored weekly for 5 weeks, and assessed at 6 months, 1 year, and 2 years after infusion. Six subjects received 2 infusions of 2 × 10 cells (low dose), and 6 subjects received 2 infusions of 8 × 10 cells (high dose). RESULTS: Mean baseline Crohn's Disease Activity Index in the low-dose and high-dose groups was 305 and 364, respectively, and mean C-reactive protein was 8 mg/L and 49 mg/L, respectively. All subjects in the low-dose group achieved a clinical response (a Crohn's Disease Activity Index decrease of ≥70 points versus baseline), and 3 achieved remission (a Crohn's Disease Activity Index decrease of ≥100 to <150 points). Two subjects in the high-dose group achieved response, and none met remission criteria. Most adverse events were mild to moderate in severity and included headache, nausea, fever, and infusion site reactions. CONCLUSIONS: PDA001 infusions appear safe and well-tolerated in subjects with treatment-resistant Crohn's disease. A response was seen in all subjects in the low-dose group. The high-dose group, with a higher baseline disease activity, had only 2 responders, suggesting a more treatment-resistant population. A phase 2 study in this patient population is ongoing.

Neuroprotective effect of human placenta-derived cell treatment of stroke in rats.

UNLABELLED: Human placenta-derived adherent (PDA001) cells are mesenchymal-like stem cells isolated from postpartum placenta. In this study, we tested whether intravenously infused PDA001 improves neurological functional recovery after stroke in rats. In addition, potential mechanisms underlying the PDA001-induced neuroprotective effect were investigated. Young adult male rats (2­3 months) were subjected to 2 h of middle cerebral artery occlusion (MCAo) and treated with PDA001 (4x10(6)) or vehicle controls [dextran vehicle or phosphate buffer saline (PBS)] via intravenous (IV) administration initiated at 4 h after MCAo. A battery of functional tests and measurements of lesion volume and apoptotic cells were performed. Immunostaining and ELISA assays for vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) and brain derived neurotrophic factor (BDNF) were performed in the ischemic brain to test the potential mechanisms underlying the neuroprotective effects of PDA001 cell treatment of stroke. PDA001 cell treatment at 4 h post stroke significantly improved functional outcome and significantly decreased lesion volume, TUNEL, and cleaved caspase 3-positive cell number in the ischemic brain, compared to MCAo-vehicle and MCAo-PBS control. Treatment of stroke with PDA001 cells also significantly increased HGF and VEGF expression in the ischemic border zone (IBZ) compared to controls. Using ELISA assays, treatment of stroke with PDA001 cells significantly increased VEGF, HGF, and BDNF levels in the ischemic brain compared to controls. CONCLUSION: When administered intravenously at 4 h after MCAo, PDA001 cells promoted neuroprotective effects. These effects induced by PDA001 cell treatment may be related to the increase of VEGF, HGF, and BDNF expression,and a decrease of apoptosis. PDA001 cells may provide a viable cell source to treat stroke.

Human placenta-derived adherent cells prevent bone loss, stimulate bone formation, and suppress growth of multiple myeloma in bone.

Human placenta has emerged as a valuable source of transplantable cells of mesenchymal and hematopoietic origin for multiple cytotherapeutic purposes, including enhanced engraftment of hematopoietic stem cells, modulation of inflammation, bone repair, and cancer. Placenta-derived adherent cells (PDACs) are mesenchymal-like stem cells isolated from postpartum human placenta. Multiple myeloma is closely associated with induction of bone disease and large lytic lesions, which are often not repaired and are usually the sites of relapses. We evaluated the antimyeloma therapeutic potential, in vivo survival, and trafficking of PDACs in the severe combined immunodeficiency (SCID)-rab model of medullary myeloma-associated bone loss. Intrabone injection of PDACs into nonmyelomatous and myelomatous implanted bone in SCID-rab mice promoted bone formation by stimulating endogenous osteoblastogenesis, and most PDACs disappeared from bone within 4 weeks. PDACs inhibitory effects on myeloma bone disease and tumor growth were dose-dependent and comparable with those of fetal human mesenchymal stem cells (MSCs). Intrabone, but not subcutaneous, engraftment of PDACs inhibited bone disease and tumor growth in SCID-rab mice. Intratumor injection of PDACs had no effect on subcutaneous growth of myeloma cells. A small number of intravenously injected PDACs trafficked into myelomatous bone. Myeloma cell growth rate in vitro was lower in coculture with PDACs than with MSCs from human fetal bone or myeloma patients. PDACs also promoted apoptosis in osteoclast precursors and inhibited their differentiation. This study suggests that altering the bone marrow microenvironment with PDAC cytotherapy attenuates growth of myeloma and that PDAC cytotherapy is a promising therapeutic approach for myeloma osteolysis.

The anti-cancer drug lenalidomide inhibits angiogenesis and metastasis via multiple inhibitory effects on endothelial cell function in normoxic and hypoxic conditions.

Lenalidomide (Revlimid) is approved for the treatment of transfusion-dependent patients with anemia due to low- or intermediate-1-risk Myelodysplastic Syndromes (MDS) associated with a del 5q cytogenetic abnormality with or without additional cytogenetic abnormalities, and in combination with dexamethasone for the treatment of multiple myeloma patients who have received at least one prior therapy. Previous reports suggest that lenalidomide is anti-angiogenic and this property appears to be related to efficacy in patients with MDS. We have investigated the effect of lenalidomide on the formation of microvessels in a novel in vitro angiogenesis assay utilizing human umbilical arterial rings and in a capillary-like cord formation assay using cultured primary endothelial cells. We found that lenalidomide consistently inhibits both sprout formation by arterial rings and cord formation by endothelial cells in a dose-dependent manner. We also found an inhibitory effect of lenalidomide on the associations between cadherin 5, beta-catenin and CD31, adherens junction proteins whose interaction is critical for endothelial cell cord formation. Furthermore, lenalidomide inhibited VEGF-induced PI3K-Akt pathway signaling, which is known to regulate adherens junction formation. We also found a strong inhibitory effect of lenalidomide on hypoxia-induced endothelial cell formation of cords and HIF-1 alpha expression, the main mediator of hypoxia-mediated effects and a key driver of angiogenesis and metastasis. Anti-metastatic activity of lenalidomide in vivo was confirmed in the B16-F10 mouse melanoma model by a >40% reduction in melanoma lung colony counts versus untreated mice. Our results suggest that inhibitory effects on microvessel formation, in particular adherens junction formation and inhibition of hypoxia-induced processes support a potential anti-angiogenic and anti-metastatic mechanism for this clinically active drug.