Normal MSCs Research Articles

The activity of tertiary lymphoid structures in high grade serous ovarian cancer is governed by site, stroma, and cellular interactions.

Most high grade serous ovarian cancers (HGSOC) originate in the fallopian tube but spread to the ovary and peritoneal cavity, highlighting the need to understand antitumor immunity across HGSOC sites. Using spatial analyses, we discover that tertiary lymphoid structures (TLSs) within ovarian tumors are less developed compared with TLSs in fallopian tube or omental tumors. We reveal transcriptional differences across a spectrum of lymphoid structures, demonstrating that immune cell activity increases when residing in more developed TLSs and produce a prognostic, spatially derived TLS signature from HGSOC tumors. We interrogate TLS-adjacent stroma and assess how normal mesenchymal stem cells MSCs (nMSCs) may support B cell function and TLS, contrary to cancer-educated MSCs (CA-MSCs) which negate the prognostic benefit of our TLS signature, suggesting that pro-tumorigenic stroma could limit TLS formation.

Alpha terpineol preconditioning enhances regenerative potential of mesenchymal stem cells in full thickness acid burn wounds

Regeneration of full thickness burn wounds is a significant clinical challenge. Direct stem cell transplantation at the wound site has a promising effect on wound regeneration. However, stem cell survival within the harsh wound environment is critically compromised. In this regard, preconditioning of stem cells with cytoprotective compounds can improve the efficiency of transplanted cells. This study evaluated the possible effect of alpha terpineol (αT) preconditioned mesenchymal stem cells (αT-MSCs) in full thickness acid burn wound. An optimized concentration of 10 μM αT was used for MSC preconditioning, followed by scratch assay analysis. A novel rat model of full thickness acid burn wound was developed and characterized via macroscopic and histological examinations. Treatment (normal and αT-MSCs) was given after 48 h of burn wound induction, and the healing pattern was examined till day 40. Skin tissues were harvested at the early (day 10) and late (day 40) wound healing phases and examined by histological grading, neovascularization, and gene expression profiling of healing mediators. In scratch assay, αT-MSCs exhibited enhanced cell migration and wound closure (scratch gap) compared to normal MSCs. In vivo findings revealed enhanced regeneration in the wound treated with αT-MSCs compared to normal MSCs and untreated control. Histology revealed enhanced collagen deposition with regenerated skin layers in normal MSC- and αT-MSC treated groups compared to the untreated control. These findings were correlated with enhanced expression of α-SMA as shown by immunohistochemistry. Additionally, αT-MSC group showed reduced inflammation and oxidative stress, and enhanced regeneration, as witnessed by a decrease in IL-1β, IL-6, TNF-α, and Bax and an increase in BCL-2, PRDX-4, GPX-7,SOD-1, VEGF, EGF, FGF, MMP-9, PDGF, and TGF-β gene expression levels at early and late phases, respectively. Overall findings demonstrated that αT exerts its therapeutic effect by mitigating excessive inflammation and oxidative stress while concurrently enhancing neovascularization. Thus, this study offers new perspectives on managing full thickness acid burn wounds in future clinical settings.

VP.62 DUX4 expression turn on the myogenic program in MSC

Muscle tissue degeneration is accompanied by fibrosis in patients with the facioscapulohumeral dystrophy (FSHD). In skeletal muscles, MSCs are present and they can participate in muscles regeneration. When a muscle is injured, MSCs can migrate towards damaged site from blood circulation and neighboring intact tissues and participate in the recovery process also. Since FSHD is a genetic disease associated with aberrant <i>DUX4</i> expression, we hypothesized that MSC involved in the regeneration process may also have overexpressed level of <i>DUX4</i>, and created a model of MSC with inducible <i>DUX4</i> expression (MSC-DUX4) using the Jagannathan's approach and construct pCW57.1-DUX4-WT from Addgen. MSCs were obtained from the Cell culture collection of the IDB RAS. <i>DUX4</i> expression was confirmed in MSC-DUX4 cells by immunofluorescent staining. We conducted a comparative analysis of the differentiation potencies of MSCs and MSCs with inducible <i>DUX4</i> expression (MSCs were from the same donor). It was shown that MSCs with induced <i>DUX4</i> expression have an increased potential for myogenic differentiation, which is not characteristic of normal MSCs. These cells can fuse and form mature myotubes expressing the main markers of late myogenesis (Troponin T, MF20). At the same time, MSCs with induced expression of <i>DUX4</i> have reduced potencies for main MSC differentiations - adipogenic and osteogenic, that was shown using biochemical, immunocytochemical and RT-PCR assays. Thus, upon induction of DUX4 expression, MSCs turn on the myogenic program, while losing the capacity for their main differentiations. This does not seem so counterintuitive, since the genes involved in myogenesis are <i>DUX4</i>-downstream target genes. However, the real contribution of MSC-DUX4 to the regeneration of muscles affected by FSHD remains to be analyzed.

MSC-Derived Extracellular Vesicles in Tumors and Therapy.

Simple SummaryTherapeutic functions of mesenchymal stroma-/stem-like cells (MSCs) are mediated predominantly through paracrine effects by the release of various different components. Upon recruitment of MSCs to damaged tissue sites or tumors, several bioactive molecules and organelles that are secreted by MSCs among others are cytokines, chemokines, metabolites, and extracellular vesicles including exosomes. The MSC-mediated cargo of released exosomes contains specific proteins and nucleic acids with varying regulatory microRNAs according to the tissue origin and the MSC microenvironment. These MSC-released exosomes are taken up by different target cells in damaged tissues to promote a regulatory network of tissue repair, including immune modulation and induction of angiogenesis. Conversely, in tumors, MSC-derived exosomes can confer predominant signals to suppress neovascularization and to relay further tumor-inhibitory effects. However, MSCs that adapted to the tumor tissue by mutual interaction with cancer cells progressively alter to an aberrant phenotype with the release of exosomes carrying tumor-supportive material.Exosomes derived from mesenchymal stroma-/stem-like cells (MSCs) as part of extracellular vesicles are considered cell-free biocompatible nanovesicles that promote repair activities of damaged tissues or organs by exhibiting low immunogenic and cytotoxic effects. Contributions to regenerative activities include wound healing, maintenance of stem cell niches, beneficial regenerative effects in various diseases, and reduction of senescence. However, the mode of action in MSC-derived exosomes strongly depends on the biological content like different regulatory microRNAs that are determined by the tissue origin of MSCs. In tumors, MSCs use indirect and direct pathways in a communication network to interact with cancer cells. This leads to mutual functional changes with the acquisition of an aberrant tumor-associated MSC phenotype accompanied by altered cargo in the exosomes. Consequently, MSC-derived exosomes either from normal tissue-originating MSCs or from aberrant tumor-associated MSCs can confer different actions on tumor development. These processes exhibiting tumor-inhibitory and tumor-supportive effects with a focus on exosome microRNA content will be discriminated and discussed within this review.

Patient Heterogeneity in Acute Myeloid Leukemia: Leukemic Cell Communication by Release of Soluble Mediators and Its Effects on Mesenchymal Stem Cells.

Acute myeloid leukemia (AML) is an aggressive bone marrow malignancy, and non-leukemic stromal cells (including mesenchymal stem cells, MSCs) are involved in leukemogenesis and show AML-supporting effects. We investigated how constitutive extracellular mediator release by primary human AML cells alters proteomic profiles of normal bone marrow MSCs. An average of 6814 proteins (range 6493−6918 proteins) were quantified for 41 MSC cultures supplemented with AML-cell conditioned medium, whereas an average of 6715 proteins (range 6703−6722) were quantified for untreated control MSCs. The AML effect on global MSC proteomic profiles varied between patients. Hierarchical clustering analysis identified 10 patients (5/10 secondary AML) showing more extensive AML-effects on the MSC proteome, whereas the other 31 patients clustered together with the untreated control MSCs and showed less extensive AML-induced effects. These two patient subsets differed especially with regard to MSC levels of extracellular matrix and mitochondrial/metabolic regulatory proteins. Less than 10% of MSC proteins were significantly altered by the exposure to AML-conditioned media; 301 proteins could only be quantified after exposure to conditioned medium and 201 additional proteins were significantly altered compared with the levels in control samples (153 increased, 48 decreased). The AML-modulated MSC proteins formed several interacting networks mainly reflecting intracellular organellar structure/trafficking but also extracellular matrix/cytokine signaling, and a single small network reflecting altered DNA replication. Our results suggest that targeting of intracellular trafficking and/or intercellular communication is a possible therapeutic strategy in AML.

P-069: Inflammasome-primed neutrophils maintain a pro-tumor microenvironment in Multiple Myeloma

<h3>Background</h3> Multiple myeloma (MM) disease progression is influenced by signals from the bone marrow (BM) microenvironment. Recently, we showed that the MM BM is characterized by inflammatory mesenchymal stromal cells (iMSCs) that transcribe MM survival factors and are predicted to recruit proliferating myeloma cells via CCL2-CCR2 interactions (de Jong et al. Nat Immunol. 2021). iMSCs also transcribed high levels of chemokines that can bind to CXCR1 and 2. Myeloid cells are known to express CXCR1/2, and have been implicated in both pro- and anti-tumor responses in various malignancies. Therefore, we hypothesized that iMSCs attract and influence myeloid populations in the MM BM. <h3>Methods</h3> Using flow cytometry, we verified expression of CXCR1/2 on myeloid cell populations in the BM of 5 newly diagnosed MM (NDMM) patients. <h3>Results</h3> CD15+ neutrophils were the most dominant population expressing these receptors, as 22.4% (± 9.8%) of cells expressed CXCR2 alone, and 72.6% (± 8.0%) expressed both CXCR1 and CXCR2. CD14+ monocytes only expressed CXCR2 (86.9% ± 15.8%). Importantly, less than 1% of myeloma cells expressed these receptors (n = 17 NDMM). As these findings suggested neutrophils as a potential target of iMSC-mediated chemotaxis, we set out to identify MM-associated alterations in this population by performing single cell RNA sequencing of the full neutrophilic lineage (n = 5 NDMM and 2 controls). Interestingly, CXCR1 and CXCR2 transcription was increased in mature neutrophils of MM patients compared to controls. Additionally, mature neutrophils of MM patients had an activated transcriptome as defined by increased transcription of C3AR1, SLPI, and IL6R, the plasma cell supportive factor TNFSF13B (encoding BAFF), and the inflammatory cytokines IL1B and IL18. Transcription of IL1B and IL18 can be regulated by pattern-recognition receptors (PRRs) binding damage-associated molecular patterns (DAMPs) resulting from e.g. matrix breakdown. Transcription of PRRs as TLR1, 2 and 4 was increased in mature neutrophils of MM patients compared to controls. Secretion of IL-1 β and IL-18 relies on the cleavage of pro-forms of these cytokines by the inflammasome, a multiprotein complex that is assembled in response to alarmins. Transcription of inflammasome components PYCARD, NLRP3 and CASP1 was increased in mature neutrophils of patients with MM. Additionally, protein levels of both IL-18 and IL-1 β are increased in BM plasma from MM patients, implicating activated neutrophils as a potential source of these cytokines. <h3>Conclusion</h3> In MM, mature neutrophils are activated and might interact with iMSCs via CXCR1/2. Moreover, neutrophils are inflammasome-primed and are likely to be a source of increased IL-1 β levels in the MM BM. As IL-1 β can activate normal MSCs to become iMSC, neutrophils and iMSCs may form a feed-forward loop in which activated neutrophils contribute to a pro-MM environment by maintaining iMSC and by directly providing BAFF to tumor cells.

Abstract 2438: Genetic activation of JUN fuels the core regulatory circuitry of de-differentiated liposarcoma

Abstract Background: As a high-grade neoplasm of connective tissues, de-differentiated liposarcoma (DDLPS) is featured by genomic amplification of MDM2, CDK4 and JUN. Super-enhancer-driven master transcription factors (RUNX1, FOSL2 and MYC) establish a core feed-forward transcriptional circuit underpinning the DDLPS malignancy. However, an important question remains unanswered: how do genomic insults interact with epigenetic dysregulation in this disease? Methods: We performed ChIP-seq analysis to map loading of transcription factors and histone marks on chromatin in DDLPS cells. Functional assays following genetic manipulation of target genes were conducted to explore their disease relevance. Results: Genomic activation of JUN represents a promising prognostic marker for DDLPS. Meanwhile, JUN locus engaged excessive H3K27ac signals in both DDLPS tissues and cells, while a markedly lower level of H3K27ac deposition was observed in normal adipose tissues, adipocytes and MSCs. JUN formed a complex with FOSL2 and other co-factors in active chromatin, especially super-enhancers. Silencing of either JUN or its co-factors disrupted the core regulatory circuitry, which was accompanied by strong deleterious impacts on both cell viability and tumorigenicity. As BET bromodomain proteins dictate transcriptionally active chromatin, targeted degradation of BET proteins by PROTAC compounds exerted potent inhibitory effects on both JUN cistrome and DDLPS development. Conclusion: Our study reveals that amplification and overexpression of JUN contribute to DDLPS development. DDLPS-associated genomic abnormalities collaborate with BET proteins to fuel disease-driving core regulatory circuitry. Citation Format: Ye Chen, Pushkar Dakle, Tuan Zea Tan, Ruby Yu-Tong Lin, Mark E. Puhaindran, Sigal Gery, Liang Xu, Phillip H. Koeffler. Genetic activation of JUN fuels the core regulatory circuitry of de-differentiated liposarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2438.

Human umbilical cord-derived mesenchymal stem cells and their chondroprogenitor derivatives reduced pain and inflammation signaling and promote regeneration in a rat intervertebral disc degeneration model.

Intervertebral disc (IVD) degeneration is an asymptomatic pathophysiological condition and a strong causative factor of low back pain. There is no cure available except spinal fusion and pain management. Stem cell-based regenerative medicine is being considered as an alternative approach to treat disc diseases. The current study aimed to differentiate human umbilical cord-mesenchymal stem cells (hUC-MSCs) into chondrocyte-like cells and to elucidate their feasibility and efficacy in the degenerated IVD rat model. Chondrogenic induction medium was used to differentiate hUC-MSCs into chondroprogenitors. Rat tail IVD model was established with three consecutive coccygeal discs. qPCR was performed to quantify the molecular markers of pain and inflammation. Histological staining was performed to evaluate the degree of regeneration. Induced chondroprogenitors showed the expression of chondrogenic genes, SOX9, TGF-β1, ACAN, BMP2, and GDF5. Immunocytochemical staining showed positive expression of chondrogenic proteins SOX9, TGF-β1, TGF-β2, and Collagen 2. In in vivo study, transplanted chondroprogenitors showed better survival, homing, and distribution in IVD as compared to normal MSCs. Expression of pain and inflammatory genes at day 5 of cell transplantation modulated immune response significantly. The transplanted labeled MSCs and induced chondroprogenitors differentiated into functional nucleus pulposus (NP) cells as evident from co-localization of red (DiI) and green fluorescence for SOX9, TGF-β1, and TGF-β2. Alcian blue and H & E staining showed standard histological features, indicating better preservation of the NP structure and cellularity than degenerated discs. hUC-MSCs-derived chondroprogenitors showed better regeneration potential as compared to normal MSCs. The pain and inflammation genes were downregulated in the treated group as compared to the degenerated IVD.

Mesenchymal stromal cell extracellular vesicles rescue mitochondrial dysfunction and improve barrier integrity in clinically relevant models of ARDS.

Alveolar epithelial–capillary barrier disruption is a hallmark of acute respiratory distress syndrome (ARDS). Contribution of mitochondrial dysfunction to the compromised alveolar-capillary barrier in ARDS remains unclear. Mesenchymal stromal cells-derived extracellular vesicles (MSC-EVs) are considered as a cell-free therapy for ARDS. Mitochondrial transfer was shown to be important for the therapeutic effects of MSCs and MSC-EVs. Here we investigated the contribution of mitochondrial dysfunction to the injury of alveolar epithelial and endothelial barriers in ARDS and the ability of MSC-EVs to modulate alveolar–capillary barrier integrity through mitochondrial transfer.Primary human small airway epithelial and pulmonary microvascular endothelial cells and human precision cut lung slices (PCLSs) were stimulated with endotoxin or plasma samples from patients with ARDS and treated with MSC-EVs, barrier properties and mitochondrial functions were evaluated. Lipopolysaccharide (LPS)-injured mice were treated with MSC-EVs and degree of lung injury and mitochondrial respiration of the lung tissue were assessed.Inflammatory stimulation resulted in increased permeability coupled with pronounced mitochondrial dysfunction in both types of primary cells and PCLSs. Extracellular vesicles derived from normal MSCs restored barrier integrity and normal levels of oxidative phosphorylation while an extracellular vesicles preparation which did not contain mitochondria was not effective. In vivo, presence of mitochondria was critical for extracellular vesicles ability to reduce lung injury and restore mitochondrial respiration in the lung tissue.In the ARDS environment, MSC-EVs improve alveolar–capillary barrier properties through restoration of mitochondrial functions at least partially via mitochondrial transfer.

Abstract B55: Epigenetic reprogramming of mesenchymal stem cells by ovarian carcinoma to facilitate metastasis

Abstract Ovarian cancer, the most deadly gynecologic cancer, resides within a complex tumor microenvironment that supports cancer growth, survival, and spread. Carcinoma-associated mesenchymal stem cells (CA-MSCs) are stromal stem cells within the ovarian TME that strongly support tumor growth promoting cancer initiation, increasing the cancer stem cell pool and enhancing chemotherapy resistance. We recently demonstrated that ovarian cancer stimulation converts normal tissue-derived MSCs into CA-MSCs. The goal of this work is to investigate the mechanism of cancer-mediated CA-MSC formation. We hypothesize that ovarian cancer stimulation induces the epigenetic reprogramming of MSCs creating CA-MSCs. EPIC array DNA methylation analysis, histone mass spectrometry, and Assay for Transposase-Accessible Chromatin using sequencing (ATACseq) were performed on CA-MSCs derived from ovarian cancer patient samples and normal MSCs derived from benign fallopian tube and omentum. We demonstrate that CA-MSCs have large-scale DNA methylation differences vs. normal tissue MSCs. This DNA methylation pattern is distinct from cancer cells; however, the CA-MSC methylation pattern is represented in the DNA methylation pattern of bulk tumor from clear-cell, endometrioid, and high-grade serous cancers, indicating the importance of stromal contribution to bulk tumor analysis. DNA methylation changes were acquired during the conversion of a normal MSC into a CA-MSC and retained after CA-MSC adipocyte and fibroblast differentiation. Histone mass spectrometry and ATACseq demonstrated enrichment in repressive histone marks and overall decreased ATACseq peaks consistent with altered chromosome accessibility. Integration of DNA methylation, ATACseq, and RNAseq data demonstrates strong correlation between epigenetic changes and transcriptomic changes. Interestingly, cell:cell adhesion pathways were consistently upregulated consistent with a partial mesenchymal-to-epithelial transition (MET). This partial MET engenders CA-MSCs with enhanced tumor cell-binding capacity. Direct tumor cell:CA-MSC binding enhanced ovarian cancer migration, extravasation, and nonadherent survival in vitro and increased ovarian cancer metastasis in vivo. This partial MET may be mediated via WT1, a mediator of MET during development, as knockdown of WT1 prevented the formation of a CA-MSCs while overexpression of WT1 enhances the formation of a CA-MSC. Collectively, we demonstrate ovarian cancer stimulates the epigenetic reprogramming of normal MSCs into CA-MSCs, thus enhancing direct tumor cell binding, which facilitates ovarian cancer metastasis. Further investigation is needed to identify the molecular drivers of this reprogramming to allow the disruption of the protumorigenic TME, which may represent a powerful approach to the treatment of ovarian cancer. Citation Format: Leonard Frisbie, HuiHui Fan, Thomas Pisanic, Hui Shen, Lan Coffman. Epigenetic reprogramming of mesenchymal stem cells by ovarian carcinoma to facilitate metastasis [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr B55.

Newly Diagnosed Acute Myeloid Leukemia Patients-Derived Bone Marrow Mesenchymal Stem Cells Suppress Daunorubicin Induced HL-60 Cell Apoptosis via Modulating Caspase-3/Survivin

To explore the role of bone marrow niche in the chemotherapy resistance of patient with acute myeloid leukemia (AML), and to investigate the effects of the MSCs on the apoptosis of HL-60 cell and its underlying mechanisms. MSCs were derived from the bone marrow of newly diagnosed AML patients (AML-MSCs) and health donors(MSCs) were co-cultured with HL-60 cells respectively. The apoptosis of HL-60 cells in the presence/absence of MSCs and/or Daunorubicin were determined by flow cytometry with Annexin V/PI double staining. In addition, the morphological features of HL-60 cells were observed by Wright-Giemsa staining, and the ratio of blasts and differentiated cells were counted. Furthermore, the expressions of apoptosis-related factors including Caspase-3, Caspase-8,Caspase-9 and Survivin were detected by Western blot. The flow cytometry showed that there was no significant change in apoptosis of HL-60 cells co-cultured with MSC derived from healthy donors or AML patients. After adding Daunorubicin into different cultural systems, the apoptotic rates of HL-60, HL-60 co-cultured with normal MSCs and HL-60 co-cultured with AML-MSCs were (49.57±7.44)%, (30.72±4.05)% and (22.99±4.08)%, respectively, which showed that normal MSCs and AML-MSCs could remarkably supress Daunorubicin-induced HL-60 apoptosis, however, there was no statistically significant difference of apoptosis between HL-60 co-cultured with normal MSCs and HL-60 co-cultured with AML-MSCs. Wright-Giemsa staining showed that most of the HL-60 cells co-cultured with AML-MSCs were primitive, and cell differentiation was unusual. In AML-MSCs co-cultured group, the cell apoptosis and differentiation caused by DNR was significant decreased, and most of HL-60 cells were initial. Western blot showed that the cleavage activity of Caspase-3 of HL-60 in AML-MSCs and normal MSCs co-cultured group was decreased, compared with HL-60 in single cultured group, moreover, the decrease was significantly in AML-MSC group. Additionally, the expression of survivin in AML-MSCs and normal MSCs co-cultured group was increased, compared with that in single cultured group, and increase was significant in AML-MSCs group. MSCs can suppress Daunorubicin-induced HL-60 apoptosis via inhibiting Caspase-3 and maintaining survivin level.

Newly Diagnosed Acute Myeloid Leukemia Patients-Derived Bone Marrow Mesenchymal Stem Cells Suppress Daunorubicin Induced HL-60 Cell Apoptosis via Modulating Caspase-3/Survivin

To explore the role of bone marrow niche in the chemotherapy resistance of patient with acute myeloid leukemia (AML), and to investigate the effects of the MSCs on the apoptosis of HL-60 cell and its underlying mechanisms. MSCs were derived from the bone marrow of newly diagnosed AML patients (AML-MSCs) and health donors(MSCs) were co-cultured with HL-60 cells respectively. The apoptosis of HL-60 cells in the presence/absence of MSCs and/or Daunorubicin were determined by flow cytometry with Annexin V/PI double staining. In addition, the morphological features of HL-60 cells were observed by Wright-Giemsa staining, and the ratio of blasts and differentiated cells were counted. Furthermore, the expressions of apoptosis-related factors including Caspase-3, Caspase-8,Caspase-9 and Survivin were detected by Western blot. The flow cytometry showed that there was no significant change in apoptosis of HL-60 cells co-cultured with MSC derived from healthy donors or AML patients. After adding Daunorubicin into different cultural systems, the apoptotic rates of HL-60, HL-60 co-cultured with normal MSCs and HL-60 co-cultured with AML-MSCs were (49.57±7.44)%, (30.72±4.05)% and (22.99±4.08)%, respectively, which showed that normal MSCs and AML-MSCs could remarkably supress Daunorubicin-induced HL-60 apoptosis, however, there was no statistically significant difference of apoptosis between HL-60 co-cultured with normal MSCs and HL-60 co-cultured with AML-MSCs. Wright-Giemsa staining showed that most of the HL-60 cells co-cultured with AML-MSCs were primitive, and cell differentiation was unusual. In AML-MSCs co-cultured group, the cell apoptosis and differentiation caused by DNR was significant decreased, and most of HL-60 cells were initial. Western blot showed that the cleavage activity of Caspase-3 of HL-60 in AML-MSCs and normal MSCs co-cultured group was decreased, compared with HL-60 in single cultured group, moreover, the decrease was significantly in AML-MSC group. Additionally, the expression of survivin in AML-MSCs and normal MSCs co-cultured group was increased, compared with that in single cultured group, and increase was significant in AML-MSCs group. MSCs can suppress Daunorubicin-induced HL-60 apoptosis via inhibiting Caspase-3 and maintaining survivin level.

Abstract TMIM-066: EZH2 REGULATES THE EPIGENETIC REPROGRAMMING OF OVARIAN CANCER PROMOTING CARCINOMA-ASSOCIATED MESENCHYMAL STEM CELLS

Abstract Ovarian tumor cells reside within a complex tumor microenvironment (TME) critical to the formation and function of ovarian cancer. Carcinoma-associated mesenchymal stem cells (CA-MSCs) are stromal progenitor cells within the TME which significantly enhance ovarian tumor cell growth and chemotherapy resistance. We demonstrated that CA-MSCs arise from ovarian tumor cell mediated reprograming of normal tissue MSCs. The resulting CA-MSC phenotype has a unique and durable expression profile without acquisition of genetic mutations, suggesting epigenetic mechanisms underpin the induction of a CA-MSC. In support of this, EPIC methylation array and ATACseq data demonstrate CA-MSCs have significant global hypermethylation and overall “closed” chromatin structure compared to normal MSCs. Further, promoter methylation alterations correlate with the gene expression profile which characterize CA-MSCs. We thus sought to investigate the mechanism underlying the cancer-mediated epigenetic reprograming of CA-MSCs. The histone-lysine N-methyltransferase Enhancer of Zeste homolog 2 (EZH2) is induced early in the conversion of a CA-MSC through tumor secreted factors. EZH2 promotes transcriptional repression through histone H3 lysine 27 trimethylation (H3K27me3) leading to a closed chromatin state. EZH2 also acts as a scaffold for DNA-methyltransferases (DNMTs) thus enhancing DNA methylation. Western blotting confirmed increases in EZH2 and the EZH2 target repressive marks H3K27me3 and H2AK119ub in CA-MSCs versus MSCs. We therefore hypothesize that ovarian cancer mediated induction of EZH2 in normal tissue MSCs is critical to the formation of a CA-MSC through epigenetic reprogramming. Normal tissue MSCs derived from benign human ovary or omentum and CA-MSCs derived from high grade serous ovarian cancer patient samples were isolated as previously described. To model the tumor-mediated induction of CA-MSCs, direct co-culture of normal tissue MSCs with high grade serous cell lines CAOV3, PEO1 or OVCAR3 were used. MSC/tumor cell co-cultures were treated with the EZH2 inhibitor Tazemetostat and/or the DNMT inhibitor 5-Azacitidine. A mathematical model, referred to as the CA-MSC classifier which uses the expression of 6 genes to accurately distinguish normal MSCs from CA-MSCs, was utilized to assess the impact of EZH2 and DNMT inhibition on the formation of a CA-MSC. In this model, values closest to 1 indicate a high likelihood of being a CA-MSC (a value of &amp;gt;0.8 meets the threshold for a CA-MSC and &amp;lt;0.2 meets the threshold for a normal MSC). Pharmacologic EZH2 inhibition significantly decreased the induction of a CA-MSC phenotype in tumor cell co-culture without impacting tumor cell or CA-MSC viability. DNMT inhibition alone yielded a modest decrease in CA-MSC induction. However, the combination of EZH2 inhibition and DNMT inhibition blocked the formation of a CA-MSC with a classifier score going from 0.9 (in untreated co-cultures) to 0.1 in dual-inhibition co-cultures. Collectively, our data suggests that ovarian tumor cell mediated EZH2 induction plays a critical role in the conversion of a MSC into a CA-MSC through epigenetic reprogramming. This reprogramming can be interrupted through targeting EZH2 and DNMT. This presents a novel target to block the formation of the tumor enhancing stromal niche thus offering a potentially powerful new approach to the treatment of ovarian cancer. Citation Format: Lenard G. Frisbie, Hui Shen, and Lan Coffman. EZH2 REGULATES THE EPIGENETIC REPROGRAMMING OF OVARIAN CANCER PROMOTING CARCINOMA-ASSOCIATED MESENCHYMAL STEM CELLS [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr TMIM-066.

Effects of Bone Marrow Mesenchymal Stem Cells Derived from Patients with Newly Diagnosed Acute Myeloid Leukemia on the Cell Proliferation, Cell Cycle and Immunophenotypes of HL-60 Cells

To explore the role of bone marrow microenvironment(niche) in the development of acute myeloid leukemia (AML) and the effect of AML patients-derived MSC on the proliferation, cell cycle and immuno-phenotypes of HL-60 cells. The MSC derived from bone marrow of patients with newly diagnosed AML were isolated and co-cultured with HL-60 cells. The effect of MSC on proliferation of HL-60 cells was detected by using 3H-TdR incorporation method, the cell cycle and immunophenotypes of HL-60 cells were detected by flow cytometry. The results of 3H-TdR incorporation assay showed that both AML-MSCs and normal MSCs remarkably suppressed the HL-60 cell proliferation in a time- and dose-dependent manner. The results of cell cycle analysis demonstrated that AML MSCs and normal MSCs induced arrest of the HL-60 cells in G0/G1 phase. The results of immunophenotyping revealed that MSCs suppressed the expression of CD11a and CD154 on the surface of HL-60 cells. Moreover, AML MSCs exhibited increased inhibitory effects than that of normal MSCs. However, no remarkable effect of MSCs on CD54 expressions of HL-60 cells was observed in the current study. AML-MSCs possess effects on HL-60 cell proliferation, cell cycle and immunophenotypes similiar to normal MSCs, but exhibited increased suppressive capacity on the expression of CD11a and CD154.

Ovarian Carcinoma-Associated Mesenchymal Stem Cells Arise from Tissue-Specific Normal Stroma.

Carcinoma‐associated mesenchymal stem cells (CA‐MSCs) are critical stromal progenitor cells within the tumor microenvironment (TME). We previously demonstrated that CA‐MSCs differentially express bone morphogenetic protein family members, promote tumor cell growth, increase cancer “stemness,” and chemotherapy resistance. Here, we use RNA sequencing of normal omental MSCs and ovarian CA‐MSCs to demonstrate global changes in CA‐MSC gene expression. Using these expression profiles, we create a unique predictive algorithm to classify CA‐MSCs. Our classifier accurately distinguishes normal omental, ovary, and bone marrow MSCs from ovarian cancer CA‐MSCs. Suggesting broad applicability, the model correctly classifies pancreatic and endometrial cancer CA‐MSCs and distinguishes cancer associated fibroblasts from CA‐MSCs. Using this classifier, we definitively demonstrate ovarian CA‐MSCs arise from tumor mediated reprograming of local tissue MSCs. Although cancer cells alone cannot induce a CA‐MSC phenotype, the in vivo ovarian TME can reprogram omental or ovary MSCs to protumorigenic CA‐MSCs (classifier score of >0.96). In vitro studies suggest that both tumor secreted factors and hypoxia are critical to induce the CA‐MSC phenotype. Interestingly, although the breast cancer TME can reprogram bone marrow MSCs into CA‐MSCs, the ovarian TME cannot, demonstrating for the first time that tumor mediated CA‐MSC conversion is tissue and cancer type dependent. Together these findings (a) provide a critical tool to define CA‐MSCs and (b) highlight cancer cell influence on distinct normal tissues providing powerful insights into the mechanisms underlying cancer specific metastatic niche formation. Stem Cells 2019;37:257–269

Suspended graphene oxide nanoparticle for accelerated multilayer osteoblast attachment.

Mimicking bone tissues having layered structures is still a significant challenge because of the lack of technologies to assemble osteoblast cell types into bone structures. One of the promising and attractive materials in biomedical and different engineering fields is graphene and graphene-based nanostructures such as graphene oxide (GO) because of their unique properties. In most studies, GO was synthesized using chemical vapor deposition method, and was coated on the substrate. In this study, we proposed a simple technique for assembly of cells that facilitates the construction of osteoblast-like structures using suspended GO synthesized by graphite powder, H2 SO4 , and KMnO4 .Toxicity effects of GO on human mesenchymal stem cells (hMSCs) derived from bone marrow were analyzed. In addition to normal MSCs, toxicity effects of GO on human cancer cell line saos-2 as an abnormal cell line that possess several osteoblastic features, was examined. The attachment and expression of osteoblast cells genes were evaluated after differentiation of MSCs to osteoblast cells in presence of suspended GO by scanning electron microscopy and real time PCR. We found that the toxicity effects of GO are dose dependent and in oseogenic medium containing suspended GO the expression level of osteoblast genes osteopontin and osteocalcin and cell adhesion markers connexin were higher than control group. Interestingly, through this method GO was found to induce multilayer osteoblast cell morphology and enhance the number of cell layer. We expect that the presented method would become a highly useful approach for bone tissue engineering. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 293-303, 2018.

A Tissue Engineered Blood Vessel Model of Hutchinson-Gilford Progeria Syndrome Using Human iPSC-derived Smooth Muscle Cells

Hutchison-Gilford Progeria Syndrome (HGPS) is a rare, accelerated aging disorder caused by nuclear accumulation of progerin, an altered form of the Lamin A gene. The primary cause of death is cardiovascular disease at about 14 years. Loss and dysfunction of smooth muscle cells (SMCs) in the vasculature may cause defects associated with HGPS. Due to limitations of 2D cell culture and mouse models, there is a need to develop improved models to discover novel therapeutics. To address this need, we produced a functional three-dimensional model of HGPS that replicates an arteriole-scale tissue engineered blood vessel (TEBV) using induced pluripotent stem cell (iPSC)-derived SMCs from an HGPS patient. To isolate the effect of the HGPS iSMCs, the endothelial layer consisted of human cord blood-derived endothelial progenitor cells (hCB-EPCs) from a separate, healthy donor. TEBVs fabricated from HGPS iSMCs and hCB-EPCs show reduced vasoactivity, increased medial wall thickness, increased calcification and apoptosis relative to TEBVs fabricated from normal iSMCs or primary MSCs. Additionally, treatment of HGPS TEBVs with the proposed therapeutic Everolimus, increases HGPS TEBV vasoactivity and increases iSMC differentiation in the TEBVs. These results show the ability of this iPSC-derived TEBV to reproduce key features of HGPS and respond to drugs.

Abstract AP17: OVARIAN TUMOR STIMULATION CONVERTS NORMAL OVARY AND OMENTAL MESENCHYMAL STEM CELLS INTO TUMOR–PROMOTING CARCINOMA–ASSOCIATED MESENCHYMAL STEM CELLS

Abstract BACKGROUND: Ovarian carcinoma-associated mesenchymal stem cells (CA-MSCs) are multipotent cells which influence the creation of the ovarian tumor microenvironment. CA-MSCs strongly promote ovarian cancer growth, cancer stem cell-like cells and chemotherapy resistance. CA-MSCs lack tumor associated somatic mutations, however compared to normal MSCs, CA-MSCs have a unique expression profile which mediates their pro-tumorigenic properties. The origin of CA-MSCs is unknown. We hypothesized that CA-MSCs are derived from local tissue MSCs reprogrammed by the ovarian tumor to become pro-tumorigenic CA-MSCs. METHODS: We define a “CA-MSC-like expression profile” of 6 genes significantly and reproducibly differentially expressed between normal human MSCs (from bone marrow (BM), omentum (OM) or ovary (OV)) and patient-derived CA-MSCs. Three normal MSC populations were grown with human ovarian cancer cells and analyzed to determine if tumor stimulation can induce a CA-MSC-like expression profile. Tumor stimulated MSCs were functionally analyzed to determine if acquisition of a CA-MSC-like expression profile correlates with development of pro-tumorigenic functions. Methylation array analysis of normal MSCs versus CA-MSCs was performed to explore the role of epigenetic regulation in the formation a CA-MSC. RESULTS: Tumor stimulation of normal OV and OM MSCs but not BM MSCs yield expression changes which approximate the CA-MSC-like expression profile. In vivo tumor stimulation of normal OM MSCs most effectively induces a CA-MSC-like expression profile. The development of a CA-MSC-like expression profile correlates with gain of pro-tumorigenic functions including induction of cancer cell chemotherapy resistance, enrichment of the ovarian cancer stem cell-like pool and enhancement of tumor growth. In contrast, tumor stimulated BM MSCs--which do not develop a CA-MSC-like expression profile--fail to gain pro-tumorigenic functions. Further, methylation array analysis demonstrates significant global differences in methylation patterns of CA-MSCs versus normal MSCs, indicating that epigenetic alterations may drive the development of the CA-MSC phenotype. CONCLUSIONS: Collectively we demonstrate that normal OV and OM MSCs are capable of becoming CA-MSCs through in vivo tumor stimulation with development of a distinct CA-MSC-like expression profile with corresponding pro-tumorigenic functions. These findings highlight the critical influence of the tumor on the normal host environment, manipulating normal cells to become cancer-supporting cells. The differential capacity of OV and OM MSCs but not BM MSCs to become CA-MSCs also highlights the heterogeneity of MSCs and may factor into the intraperitoneal tropism of ovarian cancer spread. Ultimately, understanding how tumor stimulation drives the development of CA-MSCs may identify powerful targets to disrupt the formation and function of the ovarian tumor microenvironment. Citation Format: Coffman, LG and Buckanovich, RJ. OVARIAN TUMOR STIMULATION CONVERTS NORMAL OVARY AND OMENTAL MESENCHYMAL STEM CELLS INTO TUMOR–PROMOTING CARCINOMA–ASSOCIATED MESENCHYMAL STEM CELLS [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr AP17.

JAK-STAT signaling mediates the senescence of bone marrow-mesenchymal stem cells from systemic lupus erythematosus patients.

Previous studies have revealed that bone marrow-mesenchymal stem cells (BM-MSCs) from systemic lupus erythematosus (SLE) patients exhibited early signs of senescence, which may participate in the development of SLE. However, the molecular mechanisms about this phenomenon have not been fully elucidated. In the current study, we aimed to investigate whether Janus kinase (JAK)-signaling transducers and activators of transcription (STAT) signaling mediated the senescence of BM-MSCs from SLE patients. Twelve female SLE patients and healthy subjects were enrolled in the study. All BM-MSCs were isolated by density gradient centrifugation. Western blot analysis was used to test the expression of JAK-STAT signaling molecules. We observed the activity of β-gal of cells, the changes of cytoskeletal structure by F-actin staining, and the distribution of cell cycle by flow cytometry. BM-MSCs from SLE patients showed prominent features of senescence, and abnormal activation of JAK-STAT signaling transduction, high level of phosphorylated JAK2, and STAT3. After stimulation of IFN-γ in normal MSCs, JAK-STAT signaling was activated. The cell volume and the number of senescence-associated β-galactosidase (SA-β-gal) positive in SLE BM-MSCs were increased. The organization of cytoskeleton was nearly disordered. The rate of cell proliferation was decreased. AG490, the inhibitor of JAK2, and knockdown of STAT3 in BM-MSCs, could significantly reverse the senescence. In summary, our study indicated that JAK-STAT signaling pathway may play a critical role in the senescence of SLE BM-MSCs.

Abstract B60: Identification of an ovarian tumor:stromal HH:BMP4 signaling loop critical to ovarian cancer growth and chemotherapy resistance.

Abstract The ovarian tumor microenvironment is critical to ovarian cancer growth, spread, and survival. We recently identified a novel host cell component of the tumor microenvironment, human ovarian carcinoma-associated mesenchymal stem cells (CA-MSCs). CA-MSCs are of particular interest as they are multi-potent and can differentiate to create many components of the tumor microenvironment including fibroblasts, myofibroblasts and adipocytes. CA-MSCs, compared to normal MSCs, express high levels of BMP proteins and promote tumor growth by increasing numbers of cancer stem-like cells (CSCs). We now define a CA-MSC BMP4: ovarian tumor cell Hedgehog (HH) positive feedback loop critical to the cancer promoting functions of CA-MSCs. We demonstrate that CA-MSC expression of BMP4 is induced by ovarian tumor cell-secreted Hedgehog (HH). Reciprocally, CA-MSC-derived BMP4 increases ovarian tumor cell HH expression creating a positive feedback loop. Interruption of this loop with either a HH pathway inhibitor (IPI-926 or LDE-225) or BMP4 blocking antibody decreases the induction of CA-MSC-derived BMP4 and tumor-derived HH. This signaling loop is critical to the pro-tumorigenic effects of CA-MSCs as HH inhibition blocked CA-MSC-mediated tumorigenesis and reduced the number of CSCs. Additionally, HH inhibition reversed CA-MSC -induced chemotherapy resistance leading to significant cisplatin response in established platinum-resistant tumors. Collectively, we define a critical positive feedback loop between CA-MSC-derived BMP4 and ovarian tumor cell-secreted HH providing further support for clinical targeting of the HH pathway, particularly in combination with platinum-based chemotherapy, in ovarian cancer. Citation Format: Lan G. Coffman, Yunjung Choi, Karen Mclean, Marina Pasca di Magliano, Benjamin Allen, Ronald J. Buckanovich. Identification of an ovarian tumor:stromal HH:BMP4 signaling loop critical to ovarian cancer growth and chemotherapy resistance. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B60.