Stromal Cell Conditioned Medium Research Articles

Bone marrow stromal cell-conditioned medium regenerates injured sciatic nerve by increasing expression of MPZ and NGF and decreasing apoptosis.

Despite the many benefits of mesenchymal stem cell (MSC) transplantation for tissue regeneration, there are some limitations to using them, including the high costs, applying invasive procedures, the possibility of transplant rejection, and cell malignancy. This study aimed to investigate the effect of secretions of bone marrow stromal cells (BMSCs) with the cell-free strategy on damaged sciatic nerve with an emphasis on the role of apoptosis and the expression of myelin protein zero (MPZ) and nerve growth factor (NGF) proteins. BMSCs were cultured and a 25-fold concentrated conditioned medium (CM) from the cells was provided. After creating a crush injury in the left sciatic nerve of male rats, BMSCs or CM were injected into the injured site of the nerve. Four weeks later, the expression of MPZ, NGF, Bax, and Bcl-2 proteins in the sciatic nerve and histological parameters of the sciatic nerve and gastrocnemius muscle were assessed. The results demonstrated that injection of CM decreased apoptosis and increased expression of MPZ and NGF proteins, improving remyelination and regeneration of the sciatic nerve almost as much as the transplantation of the BMSCs themselves compared to the control group. The results suggest that BMSC secretions may improve remyelination and regeneration of damaged sciatic nerve by increasing the expression of MPZ and NGF and decreasing apoptosis.

A systematic review of the efficacy, safety and satisfaction of regenerative medicine treatments, including platelet-rich plasma, stromal vascular fraction and stem cell-conditioned medium for hypertrophic scars and keloids.

The primary objective of this study is to examine the efficiency of various regenerative medicine approaches, such as platelet-rich plasma, cell therapy, stromal vascular fraction, exosomes and stem cell-conditioned medium, in the process of healing hypertrophic and keloid scars. Major databases including PubMed, Scopus and Web of Science were systematically searched, and based on the content of the articles and the inclusion and exclusion criteria, eight articles were selected. Out of these eight articles, there were two non-randomized clinical trial studies (25%), one randomized, single-blinded comparative study (12.5%), one retrospective clinical observational study (12.5%) and four randomized clinical trial studies (50%). We employed EndNote X8 and Google Sheets to conduct article reviews and extract relevant data. Following the review phase, the studies underwent analysis and categorization. In all eight reviewed studies, the effectiveness of regenerative medicine in treating hypertrophic scars and keloids has been proven. Out of these studies, five (62.5%) focused on the effectiveness of platelet-rich plasma, two study (25%) examined the effectiveness of stromal vascular fraction and one study (12.5%) explored the efficacy of stem cell-conditioned medium. In two studies (25%), the treatment methods were added to standard treatment, while in six studies (75%), regenerative medicine was used as the sole treatment method and compared with standard treatment. The use of these treatment methods did not result in any serious side effects for the patients. Regenerative medicine is an effective method with minimal side effects for the treatment of hypertrophic scars and keloids. It can be used as a monotherapy or in combination with other treatment methods. However, further studies are needed to thoroughly evaluate the effectiveness of all sub-branches of this method.

Selective homing of CAR-CIK cells to the bone marrow niche enhances control of the acute myeloid leukemia burden

AbstractAcute myeloid leukemia (AML) is a hematological malignancy derived from neoplastic myeloid progenitor cells characterized by abnormal clonal proliferation and differentiation. Although novel therapeutic strategies have recently been introduced, the prognosis of AML is still unsatisfactory. So far, the efficacy of chimeric antigen receptor (CAR)–T-cell therapy in AML has been hampered by several factors, including the poor accumulation of the blood-injected cells in the leukemia bone marrow (BM) niche in which chemotherapy-resistant leukemic stem cells reside. Thus, we hypothesized that overexpression of CXCR4, whose ligand CXCL12 is highly expressed by BM stromal cells within this niche, could improve T-cell homing to the BM and consequently enhance their intimate contact with BM-resident AML cells, facilitating disease eradication. Specifically, we engineered conventional CD33.CAR–cytokine-induced killer cells (CIKs) with the wild-type (wt) CXCR4 and the variant CXCR4R334X, responsible for leukocyte sequestration in the BM of patients with warts, hypogammaglobulinemia, immunodeficiency, and myelokathexis syndrome. Overexpression of both CXCR4wt and CXCR4mut in CD33.CAR-CIKs resulted in significant improvement of chemotaxis toward recombinant CXCL12 or BM stromal cell–conditioned medium, with no observed impairment of cytotoxic potential in vitro. Moreover, CXCR4-overexpressing CD33.CAR-CIKs showed enhanced in vivo BM homing, associated with a prolonged retention for the CXCR4R334X variant. However, only CD33.CAR-CIKs coexpressing CXCR4wt but not CXCR4mut exerted a more sustained in vivo antileukemic activity and extended animal survival, suggesting a noncanonical role for CXCR4 in modulating CAR-CIK functions independent of BM homing. Taken together, these data suggest that arming CAR-CIKs with CXCR4 may represent a promising strategy for increasing their therapeutic potential for AML.

Advanced Maternal Age Differentially Affects Embryonic Tissues with the Most Severe Impact on the Developing Brain.

Advanced maternal age (AMA) poses the single greatest risk to a successful pregnancy. Apart from the impact of AMA on oocyte fitness, aged female mice often display defects in normal placentation. Placental defects in turn are tightly correlated with brain and cardiovascular abnormalities. It therefore follows that placenta, brain and heart development may be particularly susceptible to the impact of AMA. In the current study, we compared global transcriptomes of placentas, brains, hearts, and facial prominences from mid-gestation mouse conceptuses developed in young control (7-13 wks) and aging (43-50 wks) females. We find that AMA increases transcriptional heterogeneity in all tissues, but particularly in fetal brain. Importantly, even overtly normally developed embryos from older females display dramatic expression changes in neurodevelopmental genes. These transcriptomic alterations in the brain are likely induced by defects in placental development. Using trophoblast stem cells (TSCs) as a model, we show that exposure to aging uterine stromal cell-conditioned medium interferes with normal TSC proliferation and causes precocious differentiation, recapitulating many of the defects observed in placentas from aged females. These data highlight the increased risk of AMA on reproductive outcome, with neurodevelopment being the most sensitive to such early perturbations and with potential for lifelong impact.

Characteristics of the effect of exosomes isolated from donor plasma and placenta-derived mesenchymal stromal cell-conditioned medium on the paracrine secretion of human peripheral blood mononuclear cells

Extracellular small vesicles – exosomes, attract the attention of researchers as a promising tool for regulating intercellular communication. At the same time, the therapeutic effects achieved through the use of mesenchymal stem cells (MSCs) are traditionally considered by medicine as a time-tested, multi-vector strategy for the treatment of various pathologies. In particular, in addition to the application of MSCs directly, it is important to study the products of their secretion. Aim. To study characteristics of the influence of exosomes isolated from blood plasma of healthy donors and conditioned medium of placental mesenchymal stromal cell (MSC) culture on paracrine secretion of peripheral blood mononuclear cells (PBMCs) in patients with chronic heart failure (CHF) in vitro. Materials and methods. The characteristics of paracrine secretion of PBMCs in patients with CHF by the content of proteins VEGF-A, MCP-1, ICAM-1 in their culture medium were studied in two directions: under the influence of exosomes isolated from plasma of healthy donors and exosomes isolated from placental MSC culture medium. Results. Incubation of PBMCs with plasma exosomes increased VEGF-A secretion in the group of healthy donors by 2.73 times (P ≤ 0.05), in patients with CHF – by 2 times (P ≤ 0.05); but there were a multidirectional effect on the content of ICAM-1 protein: it was 1.8 times (P ≤ 0.05) increased in the group of donors and 1.4 times (P ≤ 0.05) decreased in the group of CHF patients; MCP-1 secretion was insignificantly 10 % reduced in the donor group and did not change significantly in patients. Incubation of РВМС with exosomes isolated from MSC conditioned medium did not cause significant changes in paracrine secretion of PBMCs: there was a decrease in secretion of VEGF-A by 25 %, ICAM-1 by 17 %, MCP-1 by 22 % in the group of healthy volunteers; secretion of these proteins was 19.7 %, 22.0 % and 25.0 % decreased, respectively, in the group of CHF patients. The effects observed in the incubation of PBMCs with exosomes isolated from blood plasma of healthy donors and conditioned medium of placental MSC culture have provided valuable information for the design of future studies in this area of cell biology. Conclusions. Our studies have demonstrated in vitro the effects of exosomes isolated from donor plasma and conditioned medium on the functional properties of human PBMCs.

Abstract LB516A: The irreversible FLT3 inhibitor FF-10101 is active against a diversity of FLT3 inhibitor resistance mechanisms

Abstract Background: FLT3 tyrosine kinase inhibitors (TKIs) such as midostaurin and gilteritinib have improved clinical outcomes for patients with FLT3-mutant acute myeloid leukemia (AML), but resistance remains a problem. FF-10101 is the first irreversible, covalent inhibitor of FLT3 and has shown activity against FLT3 F691L and D835 mutations. We evaluated FF-10101 against an expanded panel of FLT3 resistance mutations and activating mechanisms of MAPK signaling associated with FLT3 TKI resistance in AML cell lines and patient samples. Results: FF-10101 was uniformly active against a series of previously unevaluated FLT3 TKI resistant D835 mutations (sensitivity = IC50 <10X of FLT3-ITD alone in Ba/F3 cells). FF-10101 was more active against F691L than gilteritinib (8-fold vs 14-fold increase in IC50 vs. FLT3-ITD, respectively). K429E and N676K mutations associated with clinical resistance to crenolanib and midostaurin, respectively, were also both FF-10101-sensitive. G697S and Y693N mutations, previously identified by random saturation mutagenesis screening to confer resistance to gilteritinib, also conferred moderate resistance to FF-10101 while Y693C conferred a high degree of resistance to both (IC50 >50X higher than FLT3-ITD). D698N, however, was FF-10101-sensitive and gilteritinib-resistant. Using a cell-based model, we confirmed C695 as the lone residue covalently bound by FF-10101. Moreover, a mutagenesis screen for FF-10101 resistance identified only C695Y/R mutations. Structural modeling showed an aromatic π-π interaction between FF-10101 and Y693 that orients an electrophilic acrylamide portion for covalent bonding of FLT3 at C695, likely explaining resistance conferred by Y693C. Confirmatory mutagenesis experiments showed that Y693F, by recapitulating the native aromatic interaction, retained FF-10101 sensitivity while the non-aromatic Y693S mutation conferred a high degree of resistance. Finally, FF-10101 exerted generally greater cytotoxicity than gilteritinib in FLT3-ITD+ AML cell lines (MOLM-14 and MV4-11) and three FLT3-mutant AML patient samples cultured in HS5 stromal cell conditioned media that models bone marrow microenvironment (BME) induced TKI resistance. In MOLM-14s, FF-10101 more effectively suppressed rebound ERK re-activation after 24h drug exposure than gilteritinib, potentially explaining this enhanced activity. Nevertheless, MOLM-14 and MV4-11s with NRAS G12C and Q61K co-mutations exhibited significant resistance to FF-10101 and gilteritinib. Conclusions: FF-10101 is active against nearly all mutations associated with FLT3 TKI resistance except for C695 mutations (site of FF-10101 binding) and the gilteritinib/crenolanib-resistant Y693C mutation (which has yet to be identified clinically). FF-10101 potentially overcomes resistance associated with the BME but likely remains vulnerable to RAS activating mutants. In report of phase 1 study of FF-10101 in relapsed/refractory AML (NCT03194685), 8/30 evaluable patients exhibited response (27%) with five responding patients previously receiving a FLT3 TKI, including one CRi after progression on gilteritinib. These results and our findings show that FF-10101 has potential to benefit AML patients that acquire certain FLT3 TKD resistance mutations or exhibit progression with other FLT3 TKIs, including gilteritinib. Citation Format: Timothy T. Ferng, Daisuke Terada, Makoto Ando, Theodore C. Tarver, Fihr Chaudhary, Kimberly C. Lin, Aaron C. Logan, Catherine C. Smith. The irreversible FLT3 inhibitor FF-10101 is active against a diversity of FLT3 inhibitor resistance mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB516A.

Chk1 Inhibition Potently Blocks STAT3 Tyrosine705 Phosphorylation, DNA-Binding Activity, and Activation of Downstream Targets in Human Multiple Myeloma Cells.

These findings identify a heretofore unrecognized link between the Chk1 and STAT3 pathways and suggest that Chk1 pathway inhibitors warrant attention as novel and potent candidate STAT3 antagonists in myeloma.

Adipose Stromal Cell-Secretome Counteracts Profibrotic Signals From IPF Lung Matrices.

Introduction: Idiopathic pulmonary fibrosis (IPF) is a fibrotic lung disease characterized by excess deposition and altered structure of extracellular matrix (ECM) in the lungs. The fibrotic ECM is paramount in directing resident cells toward a profibrotic phenotype. Collagens, an important part of the fibrotic ECM, have been shown to be structurally different in IPF. To further understand the disease to develop better treatments, the signals from the ECM that drive fibrosis need to be identified. Adipose tissue-derived stromal cell conditioned medium (ASC-CM) has demonstrated antifibrotic effects in animal studies but has not been tested in human samples yet. In this study, the collagen structural integrity in (fibrotic) lung tissue, its interactions with fibroblasts and effects of ASC-CM treatment hereon were studied.Methods: Native and decellularized lung tissue from patients with IPF and controls were stained for denatured collagen using a collagen hybridizing peptide. Primary lung fibroblasts were seeded into decellularized matrices from IPF and control subjects and cultured for 7 days in the presence or absence of ASC-CM. Reseeded matrices were fixed, stained and analyzed for total tissue deposition and specific protein expression.Results: In both native and decellularized lung tissue, more denatured collagen was observed in IPF tissue compared to control tissue. Upon recellularization with fibroblasts, the presence of denatured collagen was equalized in IPF and control matrices, whereas total ECM was higher in IPF matrices than in the control. Treatment with ASC-CM resulted in less ECM deposition, but did not alter the levels of denatured collagen.Discussion: Our data showed that ASC-CM can inhibit fibrotic ECM-induced profibrotic behavior of fibroblasts. This process was independent of collagen structural integrity. Our findings open up new avenues for ASC-CM to be explored as treatment for IPF.

CLEC‐2 stimulates IGF‐1 secretion from podoplanin‐positive stromal cells and positively regulates erythropoiesis in mice

BackgroundErythropoiesis is a complex multistep process by which erythrocytes are produced. C‐type lectin‐like receptor 2 (CLEC‐2) is a podoplanin (PDPN) receptor almost exclusively expressed on the surface of platelets and megakaryocytes. Deletion of megakaryocyte/platelet CLEC‐2 was reported to cause anemia along with thrombocytopenia in mice. PDPN‐expressing stromal cells in the bone marrow (BM) were also reported to facilitate megakaryocyte expansion and maturation depending on the CLEC‐2/PDPN interaction. ObjectivesWe investigated how specific deletion of CLEC‐2 in megakaryocytes/platelets leads to anemia. MethodsWe used flow cytometry to analyze maturation of erythroblasts, apoptotic cell death, and cell cycle distribution. CLEC‐2 stimulated PDPN‐expressing stromal cell–conditioned medium was analyzed by cytokine array and ELISA, and co‐cultured with immature erythroblasts. Cytokine levels in serum and BM extracellular fluid were quantified by ELISA. ResultsWe observed increased apoptosis of BM erythroblasts in megakaryocyte/platelet‐specific CLEC‐2 conditional knockout (Clec1bΔPLT) mice. Moreover, PDPN‐expressing stromal cells in the BM secreted insulin‐like growth factor 1 (IGF‐1) depending on the CLEC‐2/PDPN interaction. Pretreatment with IGF‐1 receptor inhibitor increased apoptosis rate and decreased the proliferation of erythroblasts in vitro. Furthermore, in Clec1bΔPLT mice, IGF‐1 concentrations in serum and BM extracellular fluid were decreased, and IGF‐1 replacement in Clec1bΔPLT mice attenuated anemia. ConclusionsOur findings suggest that IGF‐1 secretion from PDPN‐expressing stromal cells by CLEC‐2 stimulation positively regulates erythroblasts. This novel mechanism of erythropoiesis regulation indicates that a microenvironment consisting of megakaryocytes and PDPN‐expressing stromal cells supports erythropoiesis.

Aberrantly low STAT3 and STAT5 responses are associated with poor outcome and an inflammatory gene expression signature in pediatric acute myeloid leukemia

The relapse rate for children with acute myeloid leukemia is nearly 40% despite aggressive chemotherapy and often stem cell transplant. We sought to understand how environment-induced signaling responses are associated with clinical response to treatment. We previously reported that patients whose AML cells showed low G-CSF-induced STAT3 activation had inferior event-free survival compared to patients with stronger STAT3 responses. Here, we expanded the paradigm to evaluate multiple signaling parameters induced by a more physiological stimulus. We measured STAT3, STAT5 and ERK1/2 responses to G-CSF and to stromal cell-conditioned medium for 113 patients enrolled on COG trials AAML03P1 and AAML0531. Low inducible STAT3 activity was independently associated with inferior event-free survival in multivariate analyses. For inducible STAT5 activity, those with the lowest and highest responses had inferior event-free survival, compared to patients with intermediate STAT5 responses. Using existing RNA-sequencing data, we compared gene expression profiles for patients with low inducible STAT3/5 activation with those for patients with higher inducible STAT3/5 signaling. Genes encoding hematopoietic factors and mitochondrial respiratory chain subunits were overexpressed in the low STAT3/5 response groups, implicating inflammatory and metabolic pathways as potential mechanisms of chemotherapy resistance. We validated the prognostic relevance of individual genes from the low STAT3/5 response signature in a large independent cohort of pediatric AML patients. These findings provide novel insights into interactions between AML cells and the microenvironment that are associated with treatment failure and could be targeted for therapeutic interventions.

Obesity Attenuates T-Cell Function Which Impacts the Efficacy of T-Cell Based Immunotherapies in Acute Lymphoblastic Leukemia

The incidence of obesity continues to rise with over 50% of the world's population predicted to be overweight/obese by the year 2030. The global health impact of this trend is concerning given that obesity is a risk factor for developing cancers of varying etiologies. Alarmingly, the survival outcomes for obese patients with cancer are lower than those observed in lean patients. Obesity is characterized by the accumulation of adipocytes which alters drug dynamics and impacts the function of cancer and immune cells in the tumor microenvironment. Obesity-induced immune defects are troubling given the increasing use of immunotherapy in the treatment of malignancies. Here we show that adipocyte-secreted factors upregulate immunosuppressive mechanisms on human B-cell acute lymphoblastic leukemia (B-ALL) cells, attenuate the function of endogenous T-cells, and compromise the efficacy of T-cell based immunotherapies. To study the impact of adiposity on T-cell function, CD4+ and CD8+ T-cells were purified from the spleens of C57BL/6 mice and activated with PMA/Ionomycin for 72 hours in unconditioned media (UCM), bone marrow stromal-cell conditioned media (SCM), and adipocyte-conditioned media (ACM) followed by flow cytometry analysis for surface marker expression, cytokine production, and the induction of cytolytic mediators. Interestingly, T-cells activated in ACM, but not UCM or SCM, showed an attenuated phenotype highlighted by decreased CD44 and PD-1 expression, diminished cytokine production (IFN-γ/TNF-α) and reduced induction of cytolytic mediators (granzyme B/perforin). These observations were also true in obese, relative to lean, patients with B-ALL where we found that T-cells purified from the peripheral blood mononuclear cells (PBMCs) failed to produce significant levels of TNF-α when stimulated with PMA/Ionomycin. In all, these results demonstrate that adipocyte-secreted factors directly compromise the function of endogenous T-cells, which phenocopies T-cell defects observed in obese relative to lean pediatric patients with B-ALL. We next assessed the impact of adiposity on malignant cells by culturing human B-ALL cell lines in the conditioned mediums described above and performed flow cytometric analysis to assess their surface expression of the B-cell lineage antigen CD19 and proteins that modulate immunity. In addition to being a marker for B-cells, CD19 is the primary target of the T-cell based immunotherapies Blinatumomab and CAR T-cells directed against B-ALL cells. Surprisingly, when human B-ALL cells were co-cultured with adipocytes, every cell line tested (n=6) exhibited lower surface CD19 expression with 5 out 6 reaching statistical significance. Furthermore, adipocyte-secreted factors alone were sufficient to reduce CD19 surface levels on B-ALL cells in 2 of the 6 cell lines tested. Human B-ALL cells cultured in ACM, but not UCM or ACM, also upregulated their surface expression of the immunoinhibitory proteins HVEM, PD-L1, and PD-L2. These results demonstrate that adipocytes directly induce the downregulation of CD19 on human B-ALLs and increase their immune evasive capacity. Given these observations, we hypothesized that adipocyte-secreted factors would compromise T-cell-based immunotherapies targeting CD19-expressing B-ALL cells. To this end, primary human T-cells were engineered to express a CD19-directed chimeric antigen receptor (CAR). CAR T-cells and human B-ALL cells were separately pre-treated for 24 hours in UCM, SCM or ACM followed by co-culture for cytolytic analysis using Annexin-V/PI staining. Adipocyte-secreted factors significantly inhibited CAR T-cell mediated killing of CD19-expressing B-ALL cells at 4 hours. In addition to CAR T-cells, we tested the leukemia killing efficacy of the bispecific T-cell engager, Blinatumomab. After 3 days of culture, we found that Blinatumomab significantly increased the killing capacity of endogenous T-cells with 60-80% of B-ALL cells being killed after 3 days of culture in UCM and SCM. In contrast, we found that ACM significantly compromised the efficacy Blinatumomab with only 30% of B-ALL cells being killed over 3 days when co-cultured with human T-cells. Our pre-clinical data highlights the negative impact of an adipose-rich microenvironment on T-cell function and B-ALL immunogenicity, which subsequently compromises the efficacy of multiple classes of immunotherapies targeting CD19. Disclosures No relevant conflicts of interest to declare.

Obesity-Induced Chemoresistance in B-Cell Acute Lymphoblastic Leukemia Cells Is Mediated By Reduced Chemotherapy Uptake and Interleukin-9 Signaling

Background: Since the 1970's, obesity rates in children and adolescents have more than tripled (Ogden et al., 2006; Ogden et al., 2016) which parallels the rise in annual medical cost to treat obese pediatric patients in the United States which now exceeds $10 billion dollars (Beiner et al., 2020). The increasing cost to treat obese pediatric patients is in part due to more aggressive diseases which frequently present in these patients. For example, obese pediatric patients with B-cell acute lymphoblastic leukemia (B-ALL) have inferior overall survival compared to lean patients, with obese patients having a 2.5-fold increased risk of developing an adverse event and an almost 4-fold greater risk of death compared to non-obese patients (Butturini et al., 2007; Eissa et al., 2017; Ethier et al., 2012). Adipocytes accumulate with weight gain, and emerging data suggest the these cells secrete factors which promote chemoresistance in B-ALL cells (Ehsanipour et al., 2013; Sheng et al., 2016). Given the negative impact of obesity on cancer outcomes, we sought to define how adiposity impacts B-ALL pathogenesis and chemosensitivity. Methods: We performed global cytokine profiling and ELISA analyses to identify cytokines and chemokines secreted at high levels by adipocytes and present at high concentrations in the obese relative to the lean microenvironments. We then determined how candidate cytokines/chemokines impacted the function (proliferation/ signal transduction) of murine and human B-ALL cell lines using in vitro models of adiposity and the diet-induced murine model of obesity (DIO model). These experiments were also carried out on de-identified patient samples from lean and obese pediatric patients with B-ALL which were obtained from the Aflac Leukemia and Lymphoma Biorepository. To assess the impact of bone marrow stromal cells and adipocytes on B-ALL cell chemosensitivity, we exposed human B-ALL cells to stromal cell conditioned-media (SCM) and adipocyte-conditioned media (ACM) prior to methotrexate (MTX) treatment to determine their impact on cytotoxicity (Annexin-V/PI analysis) and drug uptake (confocal microscopy using fluorescently-conjugated methotrexate MTX). Results: We made the novel finding that Interleukin-9 is elevated in adipose-rich microenvironments and induces survival pathways which promote chemoresistance. From our studies, we found that obese mice succumb significantly faster than lean mice transplanted with murine Ph+ B-ALL cells. Analysis of serum obtained from lean and obese mice revealed that circulating IL-9 levels were significantly higher in mice with high adiposity. Furthermore, we found that adipocytes secreted copious amounts of IL-9; whereas, the production of this cytokine by bone marrow stromal cells was barely detectable. Interestingly, only human B-ALL cells exposed to ACM upregulated the Interleukin-9 receptor, suggesting increased sensitivity to this cytokine when malignant cells are exposed to adipocytes. Treating human B-ALL cells with recombinant IL-9 resulted in the activation of STAT3 and NF-κB signaling pathways and significantly increased proliferation. The induction of these proliferative and survival pathways coincided with reduced sensitivity to MTX treatment, whereas B-ALL cell death was abrogated when malignant cells were exposed to high levels of IL-9. Furthermore, the uptake of fluorescently-labeled MTX in ACM-exposed human B-ALL cells was significantly decreased compared to the internalization observed in B-ALL cells cultured in unconditioned media or SCM. Conclusions:It has been previously shown that IL-9 promotes chemoresistance in diffuse large B-cell lymphoma and is associated with worse prognosis in patients with B-cell chronic lymphocytic leukemia (Chen, Lv, Li, Lu, & Wang, 2014; Lv, Feng, Ge, Lu, & Wang, 2016). To our knowledge, our studies are the first to demonstrate that adipocyte-produced IL-9 promotes B-ALL pathogenesis and chemoresistance. To further explore this relationship and potential of targeting IL-9 as a novel therapeutic option, we are conducting in vitro and in vivo experiments to assess the impact of IL-9 neutralizing antibody treatment alone and when combined with frontline chemotherapy options for B-ALL Disclosures No relevant conflicts of interest to declare.

Tumor-Resident Stromal Cells Promote Breast Cancer Invasion through Regulation of the Basal Phenotype.

Collective invasion can be led by breast cancer cells expressing basal epithelial markers, typified by keratin-14 (KRT14). We analyzed gene expression data from The Cancer Genome Atlas and demonstrated a significant correlation between a KRT14+ invasion signature and a stromal-mediated extracellular matrix (ECM) organization module. We then developed a novel coculture model of tumor organoids with autologous stromal cells. Coculture significantly increased KRT14 expression and invasion of organoids from both luminal and basal murine breast cancer models. However, stromal cell conditioned medium induced invasion but not KRT14 expression. Cancer cells released TGFβ and that signaling pathway was required for stromal cell-induced invasion and KRT14 expression. Mechanistically, TGFβ induced NOX4 expression in stromal cells and NOX4 inhibition reduced invasion and KRT14 expression. In summary, we developed a novel coculture model and revealed dynamic molecular interactions between stromal cells and cancer cells that regulate both basal gene expression and invasive behavior. IMPLICATIONS: Fibroblasts within mammary tumors can regulate the molecular phenotype and invasive behavior of breast cancer cells. VISUAL OVERVIEW: http://mcr.aacrjournals.org/content/molcanres/18/11/1615/F1.large.jpg.

A nanocomposite hydrogel delivery system for mesenchymal stromal cell secretome

BackgroundMesenchymal stromal cell conditioned medium (MSC-CM) contains a cocktail of bioactive factors that act synergistically to induce therapeutic effects. This has been clearly demonstrated by in vivo applications of MSC-CM, but the establishment of controlled delivery systems is an unmet requirement for clinical translation.MethodsWe developed a nanocomposite-hydrogel (NP-H) comprised of poly-L-lactide nanoparticles (NPs) embedded in gelatin/hyaluronic acid (Gel/HA) hydrogel as a delivery vehicle for MSC-CM. First, we optimized the culture conditions for bone marrow-derived MSCs using serum-containing medium (SCM) and serum-free medium (SFM) and characterized the corresponding CM (serum-containing conditioned medium (ScCM) and serum-free conditioned medium (SfCM), respectively) for its potency and xeno markers. Then we prepared a composite matrix followed by physiochemical characterization and functional assays were performed.ResultsNanocomposite hydrogel displayed an even distribution of NPs along with high porosity (> 60%) and swelling ratios > 1500%, while its protein release pattern corresponded to a mix of degradation and diffusion kinetics. Functional evaluation of the composites was determined using MSCs and human fibroblasts (HFFs). The cells seeded directly onto the composites displayed increasing metabolic activities over time, with ScCM-NP-H groups having maximum activity. The cells treated in vitro with 5% and 10% extracts of ScCM-NP-H and SfCM-NP-H exhibited a dose- and duration-dependent response. Cell activities reduced considerably for all groups, except 10% ScCM-NP-H, which displayed a significant increase over time.ConclusionWe observed that sustained release of MSC-CM is required to prevent dose-dependent cytotoxicity. The proposed nanocomposite hydrogel for MSC-CM delivery can open up a new array for its clinical application.

Canine mesenchymal stromal cell-conditioned medium promotes survival and neurite outgrowth of neural stem cells.

We examined the paracrine action of canine mesenchymal stromal cells (MSCs) derived from bone marrow on the survival and differentiation of neural stem cells (NSCs) in vitro. MSCs were collected from the proximal end of the diaphysis of femur of healthy beagle dogs. The 70–80% confluent MSCs were re-fed with serum-free DMEM. The MSCs were incubated for 48 hr and the supernatant was collected as the conditioned medium (MSC-CM). The survival rate of NSCs in MSC-CM was significantly greater than in the medium without MSC-CM. The percentage of differentiated neurons and neurite length in MSC-CM was also significantly higher than in the medium without MSC-CM. These results suggested that canine MSC-CM promotes stem cell survival and neural differentiation of NSCs.

Abstract 505: Adipocyte-secreted proteins induce DNA damage and apoptosis in T-cell acute lymphoblastic leukemia

Abstract T-cell acute lymphoblastic leukemia (T-ALL) accounts for 10-15% of pediatric and 20-25% of adult cases of ALL. The cure rates for patients with T-ALL is approaching 80% in children while falling to 60% in adult cases. These numbers further decline in patients with primary resistance or who relapse from their initial diseases. Given the poor outcomes for patients with T-ALL, there has been growing interest in identifying risk factors that contribute to the pathogenesis of this disease. Recently, obesity (characterized by a body mass index > 30) has been shown to reduce the survival of patients with ALL. However, a closer examination of the epidemiological data revealed that obese patients with T-ALL exhibit significant improvements in survival outcomes. These data prompt several questions including; 1) what are the specific factors in the obese microenvironment that impact the function of T-ALL, 2) how do T-leukemia cells respond to these factors, and 3) are the responses influenced by mutations commonly found in T-ALL. To address these questions, we have studied how stromal cell and adipocyte-secreted factors impact the function of human T-ALL cells. Furthermore, we sought to determine if Notch1, which is mutated in > 60% of T-ALL cases, contribute to how T-leukemia cells respond to soluble factors in the microenvironment. When human T-ALL cells were grown in vitro with RPMI, stromal cell-conditioned media (SCM), or adipocyte-conditioned media (ACM), only ACM exposure resulted in extensive cell death of T-ALL (30-80% after 3 days of culture) which occurred independently of the mutational status of Notch1 and p53. The ACM secreted factor(s) that induced cell death in T-leukemia cells was revealed to be proteinaceous in nature, given that protease treatment of ACM completely abolished ACM-mediated apoptosis in T-ALL. Cell cycle analysis of human T-ALL exposed to ACM revealed an accumulation in the sub G1 population in human T-ALL, confirming the induction of apoptosis induced by the adipocyte secretome. Although the mutation status of Notch did not impact cell death, Notch1 protein levels significantly decreased in T-leukemia cells after 3 days of culture in ACM which coincided with increased γ-H2AX activation (indicative of DNA damage). This observation was confirmed using immunofluorescent microscopy, which revealed co-localization of γ-H2AX and 53BP1 (a p53 binding protein) in ACM-exposed human T-leukemia cells. Furthermore, extensive nuclear membrane blebbing was observed in human T-ALL exposed to ACM, but not SCM or unconditioned-media. Our studies reveal that adipocyte-secreted proteins are cytotoxic to human T-leukemia cells, which is independent of the mutational status of Notch1 and p53. Given this global effect, our results suggest that the obese microenvironments presents a unique opportunity to develop novel therapeutics for treating standard and high-risk patients with T-ALL. Citation Format: Miyoung Lee, Curtis J. Henry. Adipocyte-secreted proteins induce DNA damage and apoptosis in T-cell acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 505.

The skin regeneration potential of a pro-angiogenic secretome from human skin-derived multipotent stromal cells

Multipotent stromal cells stimulate skin regeneration after acute or chronic injuries. However, many stem cell therapy protocols are limited by the elevated number of cells required and poor cell survival after transplantation. Considering that the beneficial effects of multipotent stromal cells on wound healing are typically mediated by paracrine mechanisms, we examined whether the conditioned medium from skin-derived multipotent stromal cells would be beneficial for restoring the skin structure of mice after wounding. A proteomic characterization of skin-derived multipotent stromal cell-conditioned medium was performed, and the angiogenic function of this secretome was investigated in vitro using an endothelial cell tube formation assay. We then applied the skin-derived multipotent stromal cell-conditioned medium directly to full-thickness excisional wounds or embedded it into carrageenan or poly(vinyl alcohol) hydrogels to monitor tissue regeneration in mice. Biological processes related to wound healing and angiogenesis were highlighted by the analysis of the skin-derived multipotent stromal cell secretome, and a pro-angiogenic capacity for promoting tubule-like structures was first confirmed in vitro. Skin wounds treated with skin-derived multipotent stromal cell-conditioned medium also displayed increased angiogenesis, independently of the association of the conditioned medium with hydrogels. However, improvements in wound closure and epidermis or decreased inflammatory cell presence were not observed. Hence, the use of the secretome obtained from human skin-derived multipotent stromal cells may be a potential strategy to aid the natural skin repair of full-thickness lesions mainly based on its pro-angiogenic properties.

CCR5 antagonism by maraviroc inhibits Hodgkin lymphoma microenvironment interactions and xenograft growth.

Classic Hodgkin lymphoma tumor cells express a functional CCR5 receptor, and tumor tissues express high CCL5 levels, suggesting that CCL5-CCR5 signaling is involved in tumor-microenvironment formation and tumor growth. Using the CCR5 antagonist, maraviroc, and a neutralizing anti-CCL5 antibody, we found that CCL5 secreted by classic Hodgkin lymphoma cells recruited mesenchymal stromal cells and monocytes. The “education” of mesenchymal stromal cells by tumor cell-conditioned medium enhanced mesenchymal stromal cells’ proliferation and CCL5 secretion. In turn, educated mesenchymal stromal cell-conditioned medium increased the clonogenic growth of tumor cells and monocyte migration, but these effects were reduced by maraviroc. Monocyte education by tumor cell-conditioned medium induced their growth and reprogrammed them towards immunosuppressive tumor-associated macrophages that expressed IDO and PD-L1 and secreted IL-10, CCL17 and TGF-β. Educated monocyte-conditioned medium slowed the growth of phytohemagglutinin-activated lymphocytes. Maraviroc decreased tumor cell growth and synergized with doxorubicin and brentuximab vedotin. A three-dimensional heterospheroid assay showed that maraviroc counteracted both the formation and viability of heterospheroids generated by co-cultivation of tumor cells with mesenchymal stromal cells and monocytes. In mice bearing tumor cell xenografts, maraviroc reduced tumor growth by more than 50% and inhibited monocyte accumulation, without weight loss. Finally, in classic Hodgkin lymphoma human tumor tissues, CCL5 and CD68 expression correlated positively, and patients with high CCL5 levels had poor prognosis. In conclusion, since the present challenges are to find molecules counteracting the formation of the immunosuppressive tumor microenvironment or new, less toxic drug combinations, the repurposed drug maraviroc may represent a new opportunity for classic Hodgkin lym phoma treatment.

Abstract 2130: Novel ADAM9 variant induces prostate cancer metastasis

Abstract Prostate cancer often progresses from an androgen to castration-refractory prostate cancer and metastasizes to the bone and lymph nodes. However, there is no biomarker to predict the initiation of cancer metastasis. Therefore, it is an unmet need to understand molecules which induce metastasis to replace the prediction by PSA. Previous studies by us demonstrate the elevation of ADAM9 in PCa malignant progression. However, the role of ADAM9 in the tumor microenvironment is still not fully understood. Using laser capture microdissection of prostate tumor and stromal cells, indicating the increased of ADAM9 and reactive oxygen species in tumor-associated stromal cells. These inductions correlated with the increased expression of SOD-2 in tumor-associated stromal cells. Both electron microscope analysis and conditioned medium of stromal cells demonstrated the increased of soluble ADAM9 level in the medium. ELISA analysis of patient serum confirmed the increased of ADAM9 serum levels in prostate cancer patients. PCR and sequencing analysis confirmed a novel soluble ADAM9 (sADAM9v2) with deletion of membrane domain that induces cell migration through the activation of FAK and AKT phosphorylation. Our studies demonstrated the release of sADAM9 in tumor microenvironment correlated with reactive oxygen stress and regulate prostate cancer migration and metastasis. In conclusion, our studies showed that sADAM9 release by cancer-associated stromal cells could be a potential biomarker for prostate cancer progression. Citation Format: Chia-Ling Hsieh, Shian-Ying Sung. Novel ADAM9 variant induces prostate cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2130.

Abstract 3550: Lipid rafts contribute to ligand-induced signaling and survival in pediatric AML

Abstract Pediatric acute myeloid leukemia (pAML) is an aggressive malignancy with high mortality due to therapy resistance and relapse. We previously reported that the failure of pAML cells to phosphorylate STAT3 upon G-CSF or IL-6 stimulation was associated with inferior survival. The defect in STAT3 signaling was not due to low expression of receptors or of STAT3. We hypothesized that the signaling failure seen in some pAML samples is not specific for STAT3, but rather a generalized dysfunction due to abnormal lipid raft function. Lipid rafts (LR) are highly ordered plasma membrane microdomains rich in glycosphingolipids and cholesterol. Two types of LR - flat planar and invaginated caveolar - contain different membrane proteins and serve distinct but overlapping cellular functions. Both types of LR organize receptors, downstream mediators, and repressors for efficient signaling. The role of LRs in signaling and clinical outcome is not yet established in pAML. Primary samples (n=95) from the Children's Oncology Group (COG) were stimulated with HS5 stromal cell conditioned medium (HS5CM), which contains IL-6 and dozens of physiological ligands. Basal and induced phospho-STAT3 (pSTAT3), pSTAT5, pAKT and pERK1/2 were measured by intracellular flow cytometry, and the fold change in the mean fluorescence intensity (ΔMFI) was calculated. The HS5CM-induced ΔMFI for pSTAT3 was positively correlated with that for pSTAT5 (p<0.0001, r=0.5), and pAKT (p=0.0002, r=0.4). Samples that failed to increase pSTAT3 failed to activate other pathways, suggesting a generalized signaling dysfunction. In AML cells with intact signaling, disruption of LRs with methylβcyclodextrin markedly reduced HS5CM-induced pSTATs, recapitulating the dysfunctional phenotype. LRs were assessed by immunocytochemistry in 29 primary samples with and without HS5CM stimulation. Choleratoxin B, which binds ganglioside GM-1, stained LRs. Antibodies to caveolin-1 (cav-1) marked caveolar LRs. We also stained for gp130, the signaling subunit of the IL-6 receptor. Images were captured by deconvolution fluorescence microscopy. Expression was quantified by pixel intensity for average of 30 cells per condition. Colocalization of gp130 with LR markers was determined by the Manders Overlap Coefficient (MOC). Intensities and MOCs varied between samples. The MOC of gp130 with cav-1 correlated significantly with pSTAT3 ΔMFI (p=0.02, r=0.43). MOC of gp130 with GM-1 did not correlate with pSTAT3 ΔMFI (p=0.85, r=0.03), suggesting that cav-1 facilitates IL-6/STAT3 signaling. Interestingly, patients whose samples demonstrated basal cav-1 and GM-1 intensities below the median had inferior 5-year event-free survival compared to those with intensities above the median (log-rank p=0.079 for cav-1; p=0.013 for GM-1). Our results suggest that LRs contribute to ligand-induced signaling and possibly to chemotherapy response. Further studies of LR biology in AML are warranted. Citation Format: Padmini Narayanan, Julien Dubrulle, Robert B. Gerbing, Todd A. Alonzo, Fabio Stossi, Michele S. Redell. Lipid rafts contribute to ligand-induced signaling and survival in pediatric AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3550.