Presence Of Conditioned Medium Research Articles

The effect of obesity on uterine receptivity is mediated by endometrial extracellular vesicles that control human endometrial stromal cell decidualization and trophoblast invasion

AbstractThe objectives of the present study were to determine whether obesity impacts human decidualization and the endometrial control of trophoblast invasion (both of which are required for embryo implantation) and evaluate the potential involvement of endometrial extracellular vesicles (EVs) in the regulation of these physiological processes. Using primary human cell cultures, we first demonstrated that obesity is associated with significantly lower in vitro decidualization of endometrial stromal cells (ESCs). We then showed that a trophoblastic cell line's invasive ability was greater in the presence of conditioned media from cultures of ESCs from obese women. The results of functional assays indicated that supplementation of the culture medium with EVs from nonobese women can rescue (at least in part) the defect in in vitro decidualization described in ESCs from obese women. Furthermore, exposure to endometrial EVs from obese women (vs. nonobese women) was associated with significantly greater invasive activity by HTR‐8/SVneo cells. Using mass‐spectrometry‐based quantitative proteomics, we found that EVs isolated from uterine supernatants of biopsies from obese women (vs. nonobese women) presented a molecular signature focused on cell remodelling and angiogenesis. The proteomics analysis revealed two differentially expressed proteins (fibronectin and angiotensin‐converting enzyme) that might be involved specifically in the rescue of the decidualization capacity in ESCs from obese women; both of these proteins are abundantly present in endometrial EVs from nonobese women, and both are involved in the decidualization process. In conclusion, our results provided new insights into the endometrial EVs’ pivotal role in the poor uterine receptivity observed in obese women.

Secreted Soluble Factors from Tumor-Activated Mesenchymal Stromal Cells Confer Platinum Chemoresistance to Ovarian Cancer Cells.

Ovarian cancer (OC) ranks as the second most common type of gynecological malignancy, has poor survival rates, and is frequently diagnosed at an advanced stage. Platinum-based chemotherapy, such as carboplatin, represents the standard-of-care for OC. However, toxicity and acquired resistance to therapy have proven challenging for the treatment of patients. Chemoresistance, a principal obstacle to durable response in OC patients, is attributed to alterations within the cancer cells, and it can also be mediated by the tumor microenvironment (TME). In this study, we report that conditioned medium (CM) derived from murine and human stromal cells, MS-5 and HS-5, respectively, and tumor-activated HS-5, was active in conferring platinum chemoresistance to OC cells. Moreover, CM derived from differentiated murine pre-adipocyte (3T3-L1), but not undifferentiated pre-adipocyte cells, confers platinum chemoresistance to OC cells. Interestingly, CM derived from tumor-activated HS-5 was more effective in conferring chemoresistance than was CM derived from HS-5 cells. Various OC cells exhibit variable sensitivity to CM activity. Exploring CM content revealed the enrichment of a number of soluble factors in the tumor-activated HS-5, such as soluble uPAR (SuPAR), IL-6, and hepatocyte growth factor (HGF). FDA-approved JAK inhibitors were mildly effective in restoring platinum sensitivity in two of the three OC cell lines in the presence of CM. Moreover, Crizotinib, an ALK and c-MET inhibitor, in combination with platinum, blocked HGF's ability to promote platinum resistance and to restore platinum sensitivity to OC cells. Finally, exposure to 2-hydroxyestardiol (2HE2) was effective in restoring platinum sensitivity to OC cells exposed to CM. Our results showed the significance of soluble factors found in TME in promoting platinum chemoresistance and the potential of combination therapy to restore chemosensitivity to OC cells.

Aspirin synergizes with mineral particle-coated macroporous scaffolds for bone regeneration through immunomodulation.

Rationale: Mineral particles have been widely used in bone tissue engineering scaffolds due to their osteoconductive and osteoinductive properties. Despite their benefits, mineral particles can induce undesirable inflammation and subsequent bone resorption. Aspirin (Asp) is an inexpensive and widely used anti-inflammatory drug. The goal of this study is to assess the synergistic effect of Asp and optimized mineral particle coating in macroporous scaffolds to accelerate endogenous bone regeneration and reduce bone resorption in a critical-sized bone defect model. Methods: Four commonly used mineral particles with varying composition (hydroxyapatite v.s. tricalcium phosphate) and size (nano v.s. micro) were used. Mineral particles were coated onto gelatin microribbon (µRB) scaffolds. Macrophages (Mφ) were cultured on gelatin µRB scaffolds containing various particles, and Mφ polarization was assessed using PCR and ELISA. The effect of conditioned medium from Mφ on mesenchymal stem cell (MSC) osteogenesis was also evaluated in vitro. Scaffolds containing optimized mineral particles were then combined with varying dosages of Asp to assess the effect in inducing endogenous bone regeneration using a critical-sized cranial bone defect model. In vivo characterization and in vitro cell studies were performed to elucidate the effect of tuning Asp dosage on Mφ polarization, osteoclast (OC) activity, and MSC osteogenesis. Results: Micro-sized tricalcium phosphate (mTCP) particles were identified as optimal in promoting M2 Mφ polarization and rescuing MSC-based bone formation in the presence of conditioned medium from Mφ. When implanted in vivo, incorporating Asp with mTCP-coated µRB scaffolds significantly accelerated endogenous bone formation in a dose-dependent manner. Impressively, mTCP-coated µRB scaffolds containing 20 µg Asp led to almost complete bone healing of a critical-sized cranial bone defect as early as week 2 with no subsequent bone resorption. Asp enhanced M2 Mφ polarization, decreased OC activity, and promoted MSC osteogenesis in a dosage-dependent manner in vivo. These results were further validated using in vitro cell studies. Conclusions: Here, we demonstrate Asp and mineral particle-coated microribbon scaffold provides a promising therapy for repairing critical-sized cranial bone defects via immunomodulation. The leading formulation supports rapid endogenous bone regeneration without the need for exogenous cells or growth factors, making it attractive for translation. Our results also highlight the importance of optimizing mineral particles and Asp dosage to achieve robust bone healing while avoiding bone resorption by targeting Mφ and OCs.

Abstract PR017: Stromal Netrin G1 ligand (NGL-1): A new modulator of tumorigenesis and immunosuppression in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is soon to be the second deadliest types of cancer, with a 5-year survival of only 10%. The unique features of PDAC are the expansion of cancer-associated fibroblasts (CAFs), presence of a dense fibrous stroma with high bundled collagen I and immunosuppression, representing a challenge for therapies. Ways to overcome these unique pro-tumor features will be key for the development of better therapeutic strategies for PDAC. Herein we report that, in addition to the identified ectopic expression of NGL-1 in PDAC cells, it is also expressed in the stroma (CAFs and immune cells) of PDAC patients, inversely correlating with overall survival. Stromal NGL-1 was important for tumorigenesis in vivo, as NGL-1 knockout mice (KO) orthotopically allografted with pancreatic cancer cells presented less tumor burden. Further analysis showed that the tumors from KO mice presented more CD8+ T cells and less immunosuppressive cytokines, such as TGFβ. Single cell RNAseq analysis of these tumors showed decreased expression of pro-tumor factors, such as immune checkpoint molecules in T cells and TGFβ related genes across different cellular compartments (epithelial, immune and fibroblasts). In accordance with these results, these tumors presented a limited amount of desmoplastic bundled collagen, suggestive of a TGFβ-deficient environment. In order to further dissect between the NGL-1-dependent contributions of immune vs. local stroma (e.g., CAFs) cells, we generated bone marrow chimeras and performed orthotopic injections to generate tumors. The loss of NGL-1 in each cellular compartment alone failed to phenocopy the full body loss of NGL-1, suggesting that NGL-1 in both immune cells and local stromal are important for tumorigenesis. Functionally, NGL-1 deficient CAFs failed to support the survival of starved PDAC cells in vitro, downregulated important myofibroblastic molecules such as p-smad, and produced less immunosuppressive cytokines, suggesting a role for NGL-1 in key pro-tumor features of CAFs. In fact, the fibroblastic NGL-1 dependent immunomodulatory effects were confirmed with human CD8+ T cells from healthy donors, which lost their cytotoxic profile in the presence of conditioned media (CM) from NGL-1+ CAFs, but were able to keep this profile when exposed to CM from NGL-1 deficient CAFs. Bone marrow derived macrophages from KO mice produced less pro-tumor cytokines and CD8+ T cells lacking NGL-1 proliferated more than those from wild type animals, when stimulated in vitro. Mechanistically, while immune cells and CAFs deficient in NGL-1 are both tumor suppressive, the latter can regain pro-tumor functions in response to TGFβ, explaining the need for a global modulation of NGL-1 expression for an anti-tumor effect. Finally, NGL-1 KO mice orthotopically allografted with PDAC cells responded better (smaller tumors) to chemotherapeutical regimen (FOLFIRINOX) compared to WT animals. All these results point to NGL-1 as a potential new target to modulate immunosuppression and tumorigenesis in pancreatic cancer. Citation Format: Debora Barbosa Vendramini Costa, Ralph Francescone, Janusz Franco-Barraza, Tiffany Luong, Esteban Martinez, Stephen Sykes, Nina Steele, Marina Pasca di Magliano, Dmitry Zhigarev, Charline Ogier, Huamin Wang, Igor Astsaturov, Kerry Campbell, Edna Cukierman. Stromal Netrin G1 ligand (NGL-1): A new modulator of tumorigenesis and immunosuppression in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr PR017.

A comparative study of metastatic potentials of three different cancer stem cell models

Metastasis is one of the major causes of cancer deaths. However, the mechanisms of cancer cells acquiring aggressiveness and metastatic potential are still under investigation. Although cancer stem cells (CSCs) have been suggested as tumor initiating cells responsible for cancer metastasis, no sufficiently practical models have been found because of less availability of CSCs. We have developed novel CSC models derived from mouse induced pluripotent stem cells (miPSCs) in the presence of conditioned media (CM) from different cancer cell lines. Here, we tried to establish the models of metastasis using three different CSC models, miPS-LLCev, miPS-BT549cm and miPS-Huh7cm cells. The metastatic abilities of these CSC models were evaluated by intraperitoneal injections into mice. The developed metastases were histologically analyzed and the differences in gene expression between parental and metastasized cells were assessed. The expression of CSC and stemness markers was maintained in the cells isolated from metastasis. The three types of CSCs were further analyzed by RNA-sequencing to identify the enriched cytoplasmic signaling pathways. As a result, the three CSC models exhibited different patterns of metastases while the metastasis of miPS-LLCev cells appeared the most aggressive demonstrating hepatocellular carcinoma, which developed from the inside of the liver, as well as pulmonary metastasis. Metastasis related “HIF-1 pathway” was nominated as the candidate of enriched pathway in miPS-LLCev and miPS-Huh7cm cells implying that these CSC models possessed distinguished metastatic potential. Therefore, we concluded that the CSC models developed in this study would provide good models of metastasis linked with the aggressiveness.

Microglial polarization differentially affects neuronal vulnerability to the β-amyloid protein: Modulation by melatonin

Microglial cells play a central but yet debated role in neuroinflammatory events occurring in Alzheimer’s disease (AD). We here explored how microglial features are modulated by melatonin following β-amyloid (Aβ42)-induced activation and examined the cross-talk with Aβ-challenged neuronal cells. Human microglial HMC3 cells were exposed to Aβ42 (200 nM) in the presence of melatonin (MEL; 1 μM) added since the beginning (MELco) or after a 72 h-exposure to Aβ42 (MELpost). In both conditions, MEL favored an anti-inflammatory activation and rescued SIRT1 and BDNF expression/release. Caspase-1 up-regulation and phospho-ERK induction following a prolonged exposure to Aβ42 were prevented by MEL. In addition, MEL partially restored proteasome functionality that was altered by long-term Aβ42 treatment, re-establishing both 20S and 26S chymotrypsin-like activity. Differentiated neuronal-like SH-SY5Y cells were exposed to Aβ42 (200 nM for 24 h) in basal medium or in the presence of conditioned medium (CM) collected from microglia exposed for different times to Aβ42 alone or in combination with MELco or MELpost. Aβ42 significantly reduced pre-synaptic proteins synaptophysin and VAMP2 and mean neuritic length. These effects were prevented by CM from anti-inflammatory microglia (Aβ42 for 6 h), or from MELco and MELpost microglia, but the reduction of neuritic length was not rescued when the SIRT1 inhibitor EX527 was added.In conclusion, our data add to the concept that melatonin shows a promising anti-inflammatory action on microglia that is retained even after pro-inflammatory activation, involving modulation of proteasome function and translating into neuroprotective microglial effects.

Apolipoprotein E4 Effects a Distinct Transcriptomic Profile and Dendritic Arbor Characteristics in Hippocampal Neurons Cultured in vitro.

The APOE gene is diversified by three alleles ε2, ε3, and ε4 encoding corresponding apolipoprotein (apo) E isoforms. Possession of the ε4 allele is signified by increased risks of age-related cognitive decline, Alzheimer’s disease (AD), and the rate of AD dementia progression. ApoE is secreted by astrocytes as high-density lipoprotein-like particles and these are internalized by neurons upon binding to neuron-expressed apoE receptors. ApoE isoforms differentially engage neuronal plasticity through poorly understood mechanisms. We examined here the effects of native apoE lipoproteins produced by immortalized astrocytes homozygous for ε2, ε3, and ε4 alleles on the maturation and the transcriptomic profile of primary hippocampal neurons. Control neurons were grown in the presence of conditioned media from Apoe–/– astrocytes. ApoE2 and apoE3 significantly increase the dendritic arbor branching, the combined neurite length, and the total arbor surface of the hippocampal neurons, while apoE4 fails to produce similar effects and even significantly reduces the combined neurite length compared to the control. ApoE lipoproteins show no systemic effect on dendritic spine density, yet apoE2 and apoE3 increase the mature spines fraction, while apoE4 increases the immature spine fraction. This is associated with opposing effects of apoE2 or apoE3 and apoE4 on the expression of NR1 NMDA receptor subunit and PSD95. There are 1,062 genes differentially expressed across neurons cultured in the presence of apoE lipoproteins compared to the control. KEGG enrichment and gene ontology analyses show apoE2 and apoE3 commonly activate expression of genes involved in neurite branching, and synaptic signaling. In contrast, apoE4 cultured neurons show upregulation of genes related to the glycolipid metabolism, which are involved in dendritic spine turnover, and those which are usually silent in neurons and are related to cell cycle and DNA repair. In conclusion, our work reveals that lipoprotein particles comprised of various apoE isoforms differentially regulate various neuronal arbor characteristics through interaction with neuronal transcriptome. ApoE4 produces a functionally distinct transcriptomic profile, which is associated with attenuated neuronal development. Differential regulation of neuronal transcriptome by apoE isoforms is a newly identified biological mechanism, which has both implication in the development and aging of the CNS.

Evaluating the effect of conditioned medium from endometrial stem cells on endometriosis-derived endometrial stem cells.

Endometriosis is a common, benign gynecological disease which is determined as an overspreading of endometrial tissue in exterior region of the uterine cavity. Evidence suggests that retrograde menstrual blood which contains mesenchymal stem cells with differential gene expression compared to healthy women may play a role in endometriosis creation. We aimed to identify whether the conditioned medium (CM) from menstrual blood-derived mesenchymal stem cells (MenSCs) of healthy women can affect the expression level of inflammatory and stemness genes of MenSCs from endometriosis women. Endometriosis-derived MenSCs (E-MenSCs) were treated with CM derived from healthy women’s MenSCs (non-endometriosis derived MenSCs [NE-MenSCs]). Some CD markers were analyzed by flow cytometer before and after treatment compared with NE-MenSCs, and the expression level of inflammatory and stemness genes was evaluated by real-time PCR. E-MenSCs show different morphology in vitro culture in comparison with NE-MenSCs, which were changed in the presence of CM, into a morphology more similar to normal cells and showed significant decrease expression of CD10 after CM treatment. In our results, the interleukin-1, cyclooxygenase-2, and hypoxia-inducible factor 1α as inflamaturay genes and octamer-binding transcription factor 4, NANOG, and sex determining region Y-box 2 as stemness genes showed significantly different expression level in E-MenSCs after treating with CM. Our study indicates that the expression level of some inflammatory- and stemness-related genes which have differential expression in E-MenSCs compared with NE-MenSCs, could be changed to normal status by using CM derived from NE-MenSCs.

Excess glucocorticoid exposure contributes to adipose tissue fibrosis which involves macrophage interaction with adipose precursor cells

Chronic exposure to elevated glucocorticoid levels, as seen in patients with Cushing’s syndrome, can induce adipose tissue fibrosis. Macrophages play a pivotal role in adipose tissue remodelling. We used the synthetic glucocorticoid analogue dexamethasone to address glucocorticoid effects on adipose tissue fibrosis, in particular involving macrophage to preadipocyte communication. We analysed the direct effects of dexamethasone at a supra-physiological level, 0.3 µM, on gene expression of pro-fibrotic markers in human subcutaneous adipose tissue. The effects of dexamethasone on the differentiation of human SGBS preadipocytes were assessed in the presence or absence of THP1-macrophages or macrophage-conditioned medium. We measured the expression of different pro-fibrotic factors, including α-smooth muscle actin gene (ACTA2) and protein (α-SMA). Dexamethasone increased the expression of pro-fibrotic genes, e.g. CTGF, COL6A3, FN1, in adipose tissue. Macrophages abolished preadipocyte differentiation and increased the expression of the ACTA2 gene and α-SMA protein in preadipocytes after differentiation. Exposure to dexamethasone during differentiation reduced adipogenesis in preadipocytes, and elevated the expression of pro-fibrotic genes. Moreover, dexamethasone added together with macrophages further increased ACTA2 and α-SMA expression in preadipocytes, making them more myofibroblast-like. Cells differentiated in the presence of conditioned media from macrophages pretreated with or without dexamethasone had a higher expression of profibrotic genes compared to control cells. Our data suggest that macrophages promote adipose tissue fibrosis by directly interfering with preadipocyte differentiation and stimulating gene expression of pro-fibrotic factors. Excess glucocorticoid exposure also has pro-fibrotic effect on adipose tissue, but this requires the presence of macrophages.

Abstract P5-02-05: Optimizing the differentiation of the 3T3-L1 MBX mouse cell line to mature and obese adipocytes for in vitro breast cancer studies

Abstract White adipocytes are considered an endocrine organ. Present in breast tissue, they crosstalk in a paracrine manner with breast cells, secreting signals into the tumor microenvironment to drive cancer development as well as metastasis. 3T3-L1, a mouse fibroblast cell line, is widely used as an in vitro experimental model of white adipocytes for fat cell and cancer cell interaction studies. 3T3-L1 can be differentiated from the fibroblast cell line to adipocytes using a pharmacological cocktail of dexamethasone, methyl-3-isobutyl xanthine (IBMX) and insulin. Recently, ATCC has derived a clone from 3T3-L1 (ATCC CL-173), 3T3-L1 MBX, which has been claimed to have close to 100% differentiation. This 3T3-L1 MBX cell line has been recommended as a more suitable model for obesity research. However, there are few reported results describing their optimized differentiation conditions and/or any fat-cancer cell interactions. The current study has explored and optimized the concentrations of the differentiation cocktail for 3T3-L1 cells (ATCC CL-173) to differentiate its cloned derivative 3T3-L1 MBX. In the pharmacological cocktail, rosiglitazone was added as the 4th component as a prodifferentiative agent. Furthermore, the study has documented the differentiation of 3T3-L-MBX in the presence of the above-mentioned pharmacological cocktail with fatty acid combinations (palmitic acid, oleic acid, linoleic acid), to determine its applicability as an in vitro experimental model of obese adipocytes. The optimized concentrations of fatty acids that yielded the highest cell viability of 3T3-L1 MBX was determined using MTT assay. Additionally, the optimized combination of fatty acids was confirmed using oil-red-o adipogenesis staining. The adipocytokine levels of all the above mentioned in-vitro adipogenesis phases was evaluated using a mouse adipokine array kit as well as comparing the level of adipocytokines with those of fat tissue harvested from 6 month and 12 months old obese C3Hhej mice. Lastly, the study evaluated viability of three types of breast cells (non-tumorigenic, low metastatic, and highly metastatic) in the presence of conditioned medium collected from in-vitro 3T3-L1 MBX differentiated mature and obese adipocytes using an Annexin V-FITC assay kit. The study has found 3 days differentiation cycle of 3T3-L1 MBX with the addition of specific concentration of IBMX (0.5 mM), dexamethasone (1 µM), insulin (10 µg/ml) and rosiglitazone (2 µM) to be promising for its maximum differentiation. Further, 3T3-L1 MBX differentiated in presence of fatty acid were characterized to have higher lipid accumulation and adipokine secretion than the normally differentiated adipocytes. Citation Format: Noshin Mubtasim, Dr. Lauren Gollahon. Optimizing the differentiation of the 3T3-L1 MBX mouse cell line to mature and obese adipocytes for in vitro breast cancer studies [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-02-05.

273 Melanoma cells conditioned-medium induces endothelial-to-mesenchymal transition of endothelial cells

Endothelial-to-mesenchymal transition induces CAFs formation, which promote tumor growth, immunosuppression and metastasis. It is not described in melanoma, therefore we aimed to study the ability of melanoma cells to activate EndMT of endothelial cells. Human Umbilical Vein Endothelial Cells (HUVEC) were treated with conditioned medium (CM) from SK-MEL-28 melanoma cells. Analysis of protein expression (α-SMA and vonWillebrand Factor) was conducted by Western- blot. α-SMA and vWF double-stained cells were detected by FACS. Cell functional capacities were evaluated with wound healing and cell proliferation assays. TGF-β signaling pathway was analyzed through qRT-PCR and Western blot (pSMAD/SMAD and Snail). A significant increase of α-SMA expression in HUVEC was observed after 48 hours of treatment with CM as well as a slight decrease of vWF proteic expression. These findings have been confirmed by FACS analyses: a significant increase in the percentage of double-stained cells (α-SMA/vWF) has been shown after 48 hours. After 72 hours, there was a slight decrease of vWF expression. Moreover, wound healing and cell proliferation assays exhibited enhanced migration and proliferation of endothelial cells treated with CM compared to control cells. Finally, n the presence of CM, an increase in the p-Smad/Smad ratio and Snail was observed compared to control cells while no change in tgfβ1 gene expression was noticed. These data show that SK-MEL-28 cells induce a phenotypic modification of endothelial cells towards a mesenchymal profile according to a mechanism independent of the TGF-β pathway. The maintenance of endothelial markers suggests a partial and potentially reversible transition. Increased cell migration and proliferation indicate the likely formation of CAFs. Further work is required to identify the involved mediators in this process. These mediators may constitute, in the future, potential new therapeutic targets for metastatic melanoma.

Preimplantation factor modulates trophoblastic invasion throughout the decidualization of human endometrial stromal cells

BackgroundSuccessful human embryo implantation requires the differentiation of endometrial stromal cells (ESCs) into decidual cells during a process called decidualization. ESCs express specific markers of decidualization, including prolactin, insulin-like growth factor-binding protein-1 (IGFBP-1), and connexin-43. Decidual cells also control of trophoblast invasion by secreting various factors, such as matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases. Preimplantation factor (PIF) is a recently identified, embryo-derived peptide with activities at the fetal-maternal interface. It creates a favorable pro-inflammatory environment in human endometrium and directly controls placental development by increasing the human trophoblastic cells’ ability to invade the endometrium. We hypothesized that PIF’s effects on the endometrium counteract its pro-invasive effects.MethodsWe tested sPIF effect on the expression of three decidualization markers by RT-qPCR and/or immunochemiluminescence assay. We examined sPIF effect on human ESC migration by performing an in vitro wound healing assay. We analyzed sPIF effect on endometrial control of human trophoblast invasion by performing a zymography and an invasion assay.ResultsFirstly, we found that a synthetic analog of PIF (sPIF) significantly upregulates the mRNA expression of IGFBP-1 and connexin-43, and prolactin secretion in ESCs - suggesting a pro-differentiation effect. Secondly, we showed that the HTR-8/SVneo trophoblastic cell line’s invasive ability was low in the presence of conditioned media from ESCs cultured with sPIF. Thirdly, this PIF’s anti-invasive action was associated with a specifically decrease in MMP-9 activity.ConclusionTaken as a whole, our results suggest that PIF accentuates the decidualization process and the production of endometrial factors that limit trophoblast invasion. By controlling both trophoblast and endometrial cells, PIF therefore appears to be a pivotal player in the human embryo implantation process.

Dysfunctional Keratinocytes Increase Dermal Inflammation in Systemic Sclerosis: Results From Studies Using Tissue‐Engineered Scleroderma Epidermis

Evidence suggests that keratinocyte-fibroblast interactions are abnormal in systemic sclerosis (SSc). The present study was undertaken to investigate potential epidermal dysfunction in SSc and its effects on dermal homeostasis. Epidermal equivalents (EEs) were generated using keratinocytes from 6 healthy donors and 4 individuals with SSc. Skin and EE expression of markers of proliferation, differentiation, and activation was evaluated by immunohistochemistry. The transcriptomic profile of SSc EEs and healthy donor EEs was identified by RNA sequencing. EE conditioned medium (CM) was used to stimulate fibroblasts, and their production of interleukin-6 (IL-6), IL-8, matrix metalloproteinase 1 (MMP-1), type I collagen, and fibronectin was assessed by enzyme-linked immunosorbent assay. Compared to healthy donor EEs, SSc EEs exhibited aberrant differentiation, enhanced expression of activation markers, and a lower rate of basal keratinocyte mitosis, reproducing most of the abnormalities observed in SSc epidermis. RNA sequencing analysis revealed that, compared to healthy donor EEs, SSc EEs were characterized by lower expression of homeobox gene family members and by enhanced metabolic and oxidative stress molecular pathways. EE CM enhanced fibroblast production of IL-6, IL-8, MMP-1, type I collagen, and fibronectin (P < 0.05). Except for type I collagen and fibronectin, this effect was 2-fold higher in the presence of CM generated form SSc EEs. IL-1 was responsible, at least in part, for keratinocyte-dependent fibroblast activation. SSc EEs recapitulate the in vivo characteristics of SSc epidermis, demonstrating that SSc keratinocytes have an intrinsically altered differentiation program, possibly due to the dysregulation of genes from the homeobox family. The increased metabolic and oxidative stress associated with SSc epidermis may contribute to chronic inflammation and fibrosis of the dermis.

Calcitriol in the Presence of Conditioned Media from Metastatic Breast Cancer Cells Enhances Ex Vivo Polarization of M2 Alternative Murine Bone Marrow-Derived Macrophages.

Simple SummaryIn this study, we stimulated bone marrow-derived macrophages to M0, M1, and M2 subtypes, with or without calcitriol, or with or without 4T1 (metastatic), 67NR (non-metastatic), and Eph4-Ev (normal) cell culture supernatants (CMs) to test their effect on polarization. We showed that calcitriol increased the expression of Cd206 and Spp1 mRNA and CD36, CCL2, and arginase levels for M2 macrophages and decreased Cd80 and Spp1 mRNA and IL-1, IL-6, OPN, and iNOS for M1 macrophages. 4T1 CM influenced the expression of the studied genes and proteins to a greater extent than 67NR and Eph4; the strongest effect was noted for M2 macrophages. We show that calcitriol and 4T1 CM enhance the polarization of M2 macrophages and M2 macrophages differentiated with calcitriol-stimulated migration of 4T1 and 67NR cells. We indicate that the immunosuppressive properties of calcitriol may unfavorably affect the tumor microenvironment, and supplementation with vitamin D in oncological patients may not always bring benefits.In this study, we differentiated murine bone marrow-derived macrophages (BMDMs) into M0, M1, and M2 in the presence or absence of calcitriol. Real-time PCR analysis of gene expression, FACS analysis of surface markers, and chemokine/cytokine production assays were performed. In addition, the effect of the conditioned media (CM) from murine breast cancer 4T1 (metastatic) and 67NR (non-metastatic) and Eph4-Ev (normal) cells with and without calcitriol on the polarization of M1/M2 cells was determined. We found that calcitriol enhanced the differentiation of M2 macrophages, which was manifested by increased expression of Cd206 and Spp1 mRNA and CD36, Arg, and CCL2 in M2 BMDMs and by decreased expression of Cd80 and Spp1 mRNA and IL-1, IL-6, OPN, and iNOS in M1 BMDMs. 4T1 CM showed a higher effect on the gene and protein expression in macrophages than 67NR and Eph4-Ev, with the greatest effect observed on M2 macrophages which increased their differentiation and properties characteristic of alternative macrophages. Moreover, M2 macrophages differentiated with calcitriol-stimulated migration of 4T1 and 67NR cells through fibronectin and collagen type IV, respectively. Overall, our results indicated that vitamin D supplementation may not always be beneficial, especially in relation to cancers causing excessive, pathological activation of the immune system.

Y-27632 Promotes the Repair Effect of Umbilical Cord Blood-Derived Endothelial Progenitor Cells on Corneal Endothelial Wound Healing.

To investigate the proliferation of umbilical cord blood-derived endothelial progenitor cells (UCB EPCs) and the differentiation efficiency toward corneal endothelial cell (CEC)-like cells induced by rho-associated protein kinase (ROCK) inhibitor Y-27632 and to determine the most effective strategy for repairing corneal endothelium injuries in rabbits. UCB EPCs were cultured in Endothelial Cell Growth Medium-2 (EGM-2) media or conditioned media (CM) from human CECs, with and without the addition of Y-27632. Bromo-deoxyuridine (BrdU) immunocytochemistry and cell counting kit-8 assays were used to examine the proliferation of the cells. Real-time polymerase chain reaction, western blot, and immunocytochemistry were used to detect the CEC markers. Nd:YAG laser was used to establish an appropriate endothelium injury model based on rabbit corneas. The following intracameral injections were then performed to repair the model: 100 μL Opti-MEM I reduced serum medium (model group), 2 × 105 UCB EPCs diluted in 100 μL Opti-MEM I reduced serum medium (EPC group), 100 μM Y-27632 diluted in 100 μL Opti-MEM I reduced serum medium (Y-27632 group), and 2 × 105 UCB EPCs supplemented with 100 μM Y-27632 (final volume 100 μL, EPC/Y-27632 group). The follow-up tests focused on corneal transparency, central corneal thickness, intraocular pressure, and in vivo confocal microscopy, which were performed to evaluate the healing of the wounds. Culturing UCB EPCs in CM supplemented with 10 μM Y-27632 resulted in higher proliferation rates compared with EGM-2 media and CM. There were significantly improved protein levels of Zona Occludens 1, N-cadherin, Na+-K+-ATPase α1, Na+-K+-ATPase β1, and Pax6 and improved mRNA levels of collagen type IV and VIII and AQP1. The combined intracameral injection of Y-27632 and UCB EPCs accelerated the recovery of corneal transparency, regression of corneal edema, and healing of the corneal endothelium compared with the injections of Y-27632 and UCB EPCs on their own. Y-27632 not only promotes the proliferation of UCB EPCs but also contributes to differentiation of UCB EPCs toward CECs in the presence of CM. The intracameral injection of Y-27632 itself promotes the healing of corneal endothelium wounds. On this basis, supplementing UCB EPCs with Y-27632 accelerates the healing of corneal endothelium wounds.

Tumor-associated macrophages derived from cancer stem cells

Macrophages are the most abundant immune cells in the microenvironment of solid tumors. The present study displayed histological and immunohistochemical analyses of a malignant tumor model developed from cancer stem cells (CSCs) converted from human induced pluripotent stem cells (hiPSCs) in a cancer microenvironment prepared from the conditioned medium (CM) of a pancreatic cancer cell line. We focused on the localization and the origin of tumor-associated macrophages (TAMs), To the best of our knowledge this may be the first study to suggest the potential differentiation of CSCs to TAMs.hiPSCs were converted into CSCs in the presence of CM from PK8 cells. CSCs were then transplanted in vivo and formed primary tumors. Primary cultures for these tumors were serially transplanted again to obtain secondary tumors. Secondary tumors exhibited histopathological features of malignancy. Cells derived from tumors maintained the expression of endogenous stemness markers and pancreatic CSCs markers. Simultaneously, high immunoreactivity to anti-mouse CD68, anti-human CD68, CD206 and CD11b antibodies were detected revealing that the tumor tissue derived from CSCs was enriched for macrophages which can originate from both human and mouse cells.The model of CSCs highlighted the possibility of CSCs to differentiate into TAMs.

Abstract 6012: Cancer stem cells could be responsible for the chimeras of hematopoietic cells in the cancer microenvironment

Abstract Development of cancer stem cells (CSCs) model from iPSCs for different types of cancers will help to investigate the tumorigenesis and designing new therapies applicable to CSCs. In this context, our lab has developed a novel method to generate CSCs from iPSCs under cancer microenvironment using conditioned medium from different cancer cell lines. In our previous study, BT549 cell line conditioned media was used to convert iPSCs to CSCs (miPSCs-BTcscs). Here, we investigated the potential of adherent CSCs converted from iPSCs to give hematopoietic like cells. Cancer stem cells, miPSCs-BTcscs, were obtained by conversion of miPSCs (iPS MEF-Ng-20D-17) cells in the presence of conditioned medium from breast cancer cell line BT549 cells followed by the primary culture of the tumor formed in Balb/c nude mice. The stemness and tumorigenicity of miPSCs-BTcscs cells had previously been confirmed. miPSCs-BTcscs cells were cultured on gelatin coated dish in absence of Leukemia Inhibitory Factor (LIF) and select undifferentiated cells was done using puromycin. Non-adherent cells (NACs) arising from adherent CSCs collected and their viability, the morphology and the expression of hematopoietic cells markers were investigated using Giemsa staining, flow cytometry, immunofluorescence staining, and RT-PCR. Moreover, to assess the potential of clonogenicity in vivo and the engraftment of NACs, NACs were injected into the tail vein of busulfan conditioned Balb/c nude mice. The round nucleated NACs were found to be viable, positive for Lin markers and CD34 and expressed hematopoietic markers just like homing to the bone marrow. When NACs were injected into a mouse, Wright-Giemsa staining showed that the number of white blood cells got higher than those in the control mice after 4 weeks. As conclusion, CSCs were demonstrated to have the potential to provide progenies with hematopoietic markers, morphology and homing ability to bone marrow, which could give a new insight into tumor microenvironment according to the plasticity of CSCs. Citation Format: Ghmkin Hassan, Said M Afify, Kazuki Kumon, Amira Osman, Maram H Zahra, Akimasa Seno, Masaharu Seno. Cancer stem cells could be responsible for the chimeras of hematopoietic cells in the cancer microenvironment [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6012.

Abstract 1510: Immune regulators identified in undifferentiated pleomorphic sarcoma (UPS) subpopulations

Abstract Introduction: UPS is a common subtype of soft tissue sarcoma with limited effective systemic therapies. We previously showed that a murine model of UPS has various subpopulations (SPs), characterized by distinct morphology, molecular changes, and oncogenic behaviors that likely contribute to the overall behavior of UPS. Here, we aimed to characterize interactions between UPS SPs to identify pathways important in UPS progression. Methods: We used a murine model of UPS that recapitulates the human disease. Cre recombinase drives oncogenic KRAS-G12D, P53 deletion, and expression of confetti fluorescent protein to allow for in vivo lineage tracing. Individual SPs were isolated via FACS and cultured in the presence of conditioned media (CM) from other individual SPs. Growth rate was assayed using the IncuCyte system (Essen BioScience), and cell proliferation was measured using the Click-iT EdU flow cytometry assay kit. Invasion and migration were assayed using a cell culture insert with or without Matrigel, with CM or non-CM in the lower chamber. Cells migrating/invading into the lower chamber were counted. We performed proteomics analysis on the CM from the SP that potentiated growth and invasion to define the secretome of this SP. The CCL2/CCL7/CCL8 inhibitor, bindarit, was added to CM and cell confluency was assessed as above. RNAseq data from The Cancer Genome Atlas (TCGA) were used to generate immune infiltration scores in human UPS samples. These scores were correlated with expression of lead candidates from our secretome analysis. Results: CM from one UPS SP (SP-1) increased the growth rate and invasion/migration of one other SP (SP-2). Proteomics analysis identified 233 secreted proteins in the CM from SP-1, including 37 with two-fold higher abundance compared to SP-2. Among these, several immunoregulatory proteins were identified, including serpine 1, CCL2, CCL7, CXCL1, and CX3CL1. Culture with bindarit abolished the effect of SP-1 CM on growth rate. Additionally, we found several semaphorins in the secretomes of SP-1 and SP-2, and emerging evidence suggests a key role for semaphorins in cancer progression and immune regulation. Semaphorin expression correlated with immune infiltration scores in human UPS samples. Conclusions: We identified a UPS SP that positively regulates oncogenic behaviors of other SPs through secreted factors. Additionally, several of these secreted proteins play key roles in immune regulation. Given promising activity of immunotherapy in UPS, these proteins warrant further investigation. Citation Format: Candace Lynn Haddox, Junjie Bao, Tomasa Barrientos, Benjamin A. Alman. Immune regulators identified in undifferentiated pleomorphic sarcoma (UPS) subpopulations [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1510.

Abstract B74: Investigating the role of tumor:bone microenvironment crosstalk in Ewing sarcoma progression

Abstract Background: Ewing sarcoma is the second most common pediatric bone cancer. Metastatic disease is almost always fatal, and there is currently no method to predict which patients are at risk for metastasis. Better therapies are needed to prevent and treat metastatic disease, which means the mechanisms that drive Ewing sarcoma metastasis must be better elucidated. It is becoming increasingly clear that interactions between tumor cells and the tumor microenvironment (TME) play an essential role in metastasis, but the specific mechanisms through which the TME contributes to Ewing sarcoma progression remain largely unknown. Our previous work has demonstrated that activation of canonical Wnt and TGF-β pathways in a subset of Ewing sarcoma cells induces changes in gene expression and protein secretion that are associated with enhanced metastatic engraftment, changes in the extracellular matrix (ECM), and increased angiogenesis. As bone, the primary site for Ewing sarcoma, is an excellent source of ligands for both pathways, we hypothesize that crosstalk between Wnt/TGF-β-activated tumor cells and the local bone microenvironment contributes to osteolysis and metastatic progression. Methods: Ewing sarcoma cells were treated with control or Wnt3a media +/- recombinant TGF-β1, and then the mRNA levels of target genes were measured by Q-RT-PCR. ELISA and Luminex assays were also performed to determine the levels of secreted proteins. Conditioned media collected from stimulated cells were used to treat osteoblast precursor cells, which were then assayed for effects on differentiation and function. Subcutaneous, femur, and vossicle transplant xenograft models were established in mice and are being validated. Results: We have demonstrated that in response to Wnt3a and TGF-β1, Ewing sarcoma cells upregulate expression of canonical Wnt targets (e.g., LEF1), ECM-associated genes (e.g., TNC, COL1A1, and MMP2), and the pro-osteolytic factor PTHrP. We have also shown that osteoblast precursor cells can be induced to differentiate in the presence of conditioned media from Ewing sarcoma cell lines. Immunohistochemical staining is being used to evaluate Wnt and TGF-β pathway activation, angiogenesis, osteolysis, and the ECM in our bone tumor xenograft models. Conclusions: Wnt3a/TGF-β1-stimulated Ewing sarcoma cells upregulate expression of genes associated with the ECM and osteolysis. We are in the process of demonstrating the functional consequences of those changes using in vitro conditioned media experiments and in vivo transplant models. Understanding the mechanisms by which these pathways contribute to Ewing sarcoma phenotypes is important for the development of new therapeutics for patients with metastatic disease. Citation Format: Kelsey Temprine, Sydney Treichel, Allegra Hawkins, Tahra Suhan, Wei Jiang, Parker Acevedo, Amy Koh, Kurt Hankenson, Laurie K. McCauley, Elizabeth R. Lawlor. Investigating the role of tumor:bone microenvironment crosstalk in Ewing sarcoma progression [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B74.

Altered iron metabolism in cystic fibrosis macrophages: the impact of CFTR modulators and implications for Pseudomonas aeruginosa survival

Cystic fibrosis (CF) is a genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, resulting in chronic bacterial lung infections and tissue damage. CF macrophages exhibit reduced bacterial killing and increased inflammatory signaling. Iron is elevated in the CF lung and is a critical nutrient for bacteria, including the common CF pathogen Pseudomonas aeruginosa (Pa). While macrophages are a key regulatory component of extracellular iron, iron metabolism has yet to be characterized in human CF macrophages. Secreted and total protein levels were analyzed in non-CF and F508del/F508del CF monocyte derived macrophages (MDMs) with and without clinically approved CFTR modulators ivacaftor/lumacaftor. CF macrophage transferrin receptor 1 (TfR1) was reduced with ivacaftor/lumacaftor treatment. When activated with LPS, CF macrophage expressed reduced ferroportin (Fpn). After the addition of exogenous iron, total iron was elevated in conditioned media from CF MDMs and reduced in conditioned media from ivacaftor/lumacaftor treated CF MDMs. Pa biofilm formation and viability were elevated in conditioned media from CF MDMs and biofilm formation was reduced in the presence of conditioned media from ivacaftor/lumacaftor treated CF MDMs. Defects in iron metabolism observed in this study may inform host–pathogen interactions between CF macrophages and Pa.