Explant Research Articles

Ferritin light chain deficiency-induced ferroptosis is involved in preeclampsia pathophysiology by disturbing uterine spiral artery remodelling

The proteomic analysis from samples of patients with preeclampsia (PE) displayed a low level of ferritin light chains (FTL), but we do not know what the significance of reduced FTL in PE pathophysiology is. To address this question, we first demonstrated that FTL was expressed in first- and third-trimester cytotrophoblasts, including extravillous trophoblasts (EVTs), of the human placenta. Furthermore, a pregnant rat model of FTL knockdown was successfully established by intravenously injecting adenoviruses expressing shRNA targeting FTL. In pregnant rats with downregulated FTL, we observed PE-like phenotypes and impaired spiral arterial remodelling, implying a causal relationship between FTL downregulation and PE. Blocking ferroptosis with ferrostatin-1 (Fer-1) significantly rescued the above PE-like phenotypes in pregnant rats with FTL knockdown. Furthermore, using trophoblast cell line and chorionic villous explant culture assays, we showed that FTL downregulation induced cell death, especially ferroptosis, resulting in defective uterine spiral artery remodelling. Eventually, this conclusion from the animal model was verified in PE patients’ placental tissues. Taken together, this study revealed for the first time that FTL reduction during pregnancy triggered ferroptosis and then caused defective uterine spiral artery remodelling, thereby leading to PE.

Enhancement of shoot organogenesis in Polygonum tinctorium by sucrose and gelling agents

Indigo (Polygonum tinctorium), though it is a medicinal and dye crop, can also be grown in temperate areas, especially because it is commonly disseminated in Japan. To date, much research has been considered under investigation, especially for the regeneration of the indigo plant. Here in this study, we investigated the response of sucroses and gelling agents on the shoot organogenesis of the indigo plant. Micropropagation in terms of shoot regeneration and its growth was highly responded to sucrose and gelling agents. While culturing of internode explants on initial shoot regeneration media supplemented with sucrose and gelling agents of phytagar and gelrite significantly upgraded the regeneration efficiency as well as shoot growth. The regeneration capacity of the shoot was augmented with increased levels of sucrose up to 40 g L-1 and then started to decrease, whereas the increasing pattern continued even at the highest concentration (50 g L-1). The highest shoot regeneration (6.0 ± 0.5) was achieved by the treatment of 40 g L-1 giving 7.5 times higher shoot regeneration compared to the control. The increasing pattern for shoot length was more pronounced than that of shoot regeneration. The shoot length ranged from 10.2 mm to 23.5 mm within the sucrose treatments. In this study, the highest shoot length (23.5± 0.21) was observed by the treatment of 50 g L-1 exhibiting 2.3 times higher shoot length compared to the control. Gelling agent gelrite performed better than phytagar for both regeneration and shoot length growth. The shoot regeneration among the phytagar treatments ranged from 3.8 to 6.0 shoots/explant whereas the shoot regeneration ranged from 6.6 to 7.2 among the gelrite treatments. The highest shoots/explant (7.2) and the longest shoot length (22.4 mm) were observed due to the treatment of gelrite 3. The lowest shoot regeneration and shoot length were denoted when phytagar 9 was applied. It is proposed from our study that sucrose and gelling agent especially gelrite 3 could be applied in shoot organogenesis and plant transformation of any plant species, especially for P. tinctorium.

Understanding the effects of opioids vs non-opioids in the treatment of neonatal abstinence syndrome, an in vitro model.

Neonatal abstinence syndrome (NAS) refers to cadre of withdrawal manifestations in infants born to mothers who used illicit and licit substances during pregnancy. The increasing prevalence of NAS has been largely due to the maternal use of opioids during pregnancy. NAS contributes to increased morbidity and long-term disability in surviving infants. Clinically, oral opioid therapies for opioid exposure have been a standard treatment with morphine (MO) being the most commonly used medication. Recently, a non-opioid agent, clonidine (CD) has also been used with potentially favorable short- and long-term outcomes in infants. However, data regarding the cellular and molecular effects of these treatments on the developing brain is still lacking due to a lack of a reliable animal model that targets the neonatal brain. To address this gap in knowledge we determined the effects of MO or CD on the cell death of neonatal cortical explant cultures that were exposed to oxycodone (OXY) in utero. Sprague Dawley rats were randomized and implanted with programmable infusion pumps before mating to receive either the OXY (dose increasing from 1.21-1.90 mg/kg/day to a maximum dose of 2.86-3.49 mg/kg/day) or normal saline (NS) throughout pregnancy and until one week after delivery. Male and female rat pups were sacrificed on postnatal day 4, and the prefrontal cortex (PFC) and hippocampus (HC) were dissected and treated with MO (0.10-1.00 µM) or CD (1.20-120.00 µM) in culture media. After 5 days of treatment the explants were labeled with propidium iodide to detect cell death. Dead cells were analyzed and counted under fluorescence microscopy. In explants from the PFC, cell death was greater in those prenatally exposed to OXY and postnatally treated with MO (OXY/MO) (736.8 ± 76.5) compared to OXY/CD (620.9 ± 75.0; p = 0.005). In the HC explants, mean cell death counts were not significantly different between groups regardless of prenatal exposure or postnatal treatment (p = 0.19). The PFC is vital in controlling higher-order executive functions such as behavioral flexibility, learning and working memory. Therefore, our finding is consistent with executive function problems in children with prenatal opioid exposure.

Setd5, but not Setd2, is indispensable for retinal cell survival and proliferation.

Trimethylation of histone H3 at lysine 36 (H3K36me3) is associated with active transcription. We used mouse retinal explant cultures and shRNA to investigate the roles of Setd2 and Setd5, which encode H3K36me3 methyltransferases, in retinal development. We found that shSetd5 caused abnormal retinal structures and reduced rods and Müller cells, whereas shSetd2 did not cause any abnormalities. The mutant SETD5 lacking the SET domain failed to reverse the phenotypes observed in the shSetd5-expressing retinas, while SETD5S1257*, which does not interact with HDAC3 and PAF1 complexes, rescued proliferation, but not apoptosis, induced by shSetd5. Taken together, we found that Setd5, but not Setd2, is essential for sustaining retinal cell survival and proliferation, and the SET domain of SETD5 is pivotal for both functions.

Comparison of two isolation methods of tobacco-derived extracellular vesicles, their characterization and uptake by plant and rat cells

Plant extracellular vesicles (pEVs) derived from numerous edible sources gain a lot of attention in recent years, mainly due to the potential to efficiently carry bioactive molecules into mammalian cells. In the present study, we focus on isolation of PDNVs (plant-derived nanovesicles) and pEVs from callus culture and from BY-2 culture of Nicotiana tabacum (tobacco). Tobacco was selected as a source of plant vesicles, as it is commonly used by human, moreover it is a model organism with established techniques for cultivation of explant cultures in vitro. Explant cultures are suitable for the isolation of pEVs in large quantities, due to their fast growth in sterile conditions. As the efficiency of isolation methods varies, we were comparing two methods of isolation. We evaluated biophysical and biochemical properties of plant vesicles, as well as differences between isolates. We encountered difficulties in the form of vesicles aggregation, which is often described in publications focused on mammalian nanovesicles. In an effort to prevent vesicle aggregation, we used trehalose in different stages of isolation. We show tobacco-derived vesicles successfully enter tobacco and mesenchymal cell lines. We observed that tobacco-nanovesicles isolated by different methods incorporated fluorescent dye with different efficiency. The results of our study show tobacco-derived vesicles isolated by various isolation methods are able to enter plant, as well as mammalian cells.

Viability of mitochondria-labeled retinal ganglion cells in organotypic retinal explant cultures by two methods

Retinal explant cultures provide a valuable system to study retinal function in vitro. This study established a new retinal explant culture method to prolong the survival of retinal ganglion cells (RGCs). Explants were prepared in two different ways: with or without optic nerve. Retinas from newborn mice that had received an injection of MitoTracker Red into the contralateral superior colliculus to label axonal mitochondria were cultured as organotypic culture for 7 days in vitro. At several time points during the culture, viability of RGCs was assessed by multi-electrode array recording, and morphology by immunohistochemical methods. During the culture, the thickness of the retinal tissue in both groups gradually decreased, however, the structure of the layers of the retina could be identified. Massive apoptosis in the retinal ganglion cell layer (GCL) appeared on the first day of culture, thereafter the number of apoptotic cells decreased. Glial activation was observed throughout the culture, and there was no difference in morphology between the two groups. RGCs loss was exacerbated on 3rdday of culture, and RGCs loss in retinal explants with preserved optic nerve was significantly lower than in retinas that did not preserve the optic nerve. More and longer-lasting mitochondrial signals were observed in the injured area of the optic nerve-preserving explants. Retinal explants provide an invaluable tool for studying retinal function and developing treatments for ocular diseases. The optic nerve-preserving culture helps preserve the integrity of RGCs. The higher number of mitochondria in the nerve-preserving cultures may help maintain viability of RGCs.

Low Baseline Expression of Fibrotic Genes in an Ex Vivo Human Skin Model is a Potential Indicator of Excessive Skin Scarring.

An ex vivo model of skin fibrosis was used with abdominal and breast skin tissue from 45 patients undergoing breast reduction and/or abdominoplasty. Fibrosis was induced in skin explants in organ culture with transforming growth factor-β (TFGβ). Fibrotic gene response was assessed via quantitative real-time polymerase chain reaction and correlated with skin location, age, and baseline levels of fibrotic genes. The increase in TFGβ-induced fibronectin1 (FN1) gene expression in skin explants was significantly higher than for Collagen 1A1, alpha smooth muscle actin, and connective tissue growth factor. Also, FN1 expression positively correlated with donor age. Moreover, lower expression of the fibrotic genes FN1, Collagen 1A1, and alpha smooth muscle actin correlated with a more pronounced fibrotic response, represented by higher induction levels of these genes. Skin sites exhibit different baseline levels of profibrotic genes. Further, low baseline expression levels of fibrotic genes FN1, Collagen 1A1, and alpha smooth muscle actin, in donor skin may indicate a potential for excessive scarring of the skin.

Abstract B028: Bacterial cytotoxin therapy limits tumor growth for pancreatic ductal adenocarcinoma

Abstract Treating pancreatic ductal adenocarcinoma (PDAC) with systemic chemotherapeutic drugs has remained a challenge, due in part to the hypovascularized and poorly perfused nature of PDAC tumors, impeding the accumulation of systemically delivered drugs. Several clinical trials aimed at improving drug delivery in PDAC, through targeting of ECM components (HALO-301) or stromal angiogenic signaling (IPI-926-03) have unfortunately not been effective. However, the features that have interfered with systemic therapy in PDAC are potential advantages for the use of bacterial therapies, as bacteria can actively migrate through tissues, thrive in hypoxic microenvironments, and benefit from local immune suppression. Recent developments in the field of synthetic biology have made it possible to engineer complex logic circuits into bacteria, enabling the production of anticancer therapies directly within the tumor parenchyma. Furthermore, live bacteria, once colonized within the tumor niche, are capable of providing a stable source of anticancer compounds directly, rather than relying on repeated systemic doses. We have therefore worked to develop novel bacterial strains and demonstrate preclinical efficacy of a novel strain of therapeutic bacteria for targeting PDAC. We began by testing a range of bacteria-produced toxins and identified the pore-forming protein theta toxin as having the greatest effect in both 2D cell culture and PDAC explant (tissue slice) models. We then engineered a non-toxic probiotic bacteria, E. coli Nissle 1917, to produce either theta toxin or GFP following induction with acyl-homoserine lactone (AHL). To assess preclinical efficacy, we performed intratumoral injections of live GFP- and theta-expressing bacteria into the “KPC” genetically engineered mouse model (Kras LSL.G12D/+; Tp53 LSL.R172H/+; PdxCre tg/+). While GFP-producing bacteria did not induce a change in tumor growth kinetics, treatment with theta toxin-producing bacteria demonstrated prolonged stabilization of tumor growth, increasing the doubling time from 13.7 days (GFP) to 32.5 days (theta) without additional therapy. Indeed, one theta-treated KPC animal lived 113 days following a single bacterial injection, compared to a median of ~12 days for vehicle- or gemcitabine-treated historical controls. Histological analyses demonstrated that diffuse populations of bacteria co-localized with regions of tumor necrosis and cell death, but that bacterial presence and evidence of increased cell death was not observed in healthy tissues, such as the lung, liver, intestine, and diaphragm. Strikingly, while there was minimal spread of bacteria to non-tumor tissues, we observed translocation of the bacteria to regions of liver metastases and distant papillomas following injection of the primary pancreatic tumor, suggesting a mechanism for targeting both known and unknown metastases following local administration. Together these studies demonstrate potent preclinical activity of cytotoxic bacterial therapy as a novel strategy to circumvent the challenges of systemic treatment of PDAC. Citation Format: Amanda R. Decker, Tetsuhiro Harimoto, Steve A. Sastra, Tal Danino, Kenneth Olive. Bacterial cytotoxin therapy limits tumor growth for pancreatic ductal adenocarcinoma [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 B028.

De Novo Hematopoiesis from the Fetal LUNG

Hemogenic endothelial cells (HECs) are specialized cells that undergo endothelial to hematopoietic transition (EHT) to give rise to the earliest precursors of hematopoietic progenitors that will eventually sustain hematopoiesis throughout the lifetime of an organism. Although HECs are thought to be primarily limited to the aorta gonad mesonephros (AGM) during early development, EHT has been described in various other hematopoietic organs and embryonic vessels. Though not traditionally seen as a hematopoietic organ, the lung houses many resident hematopoietic cells, aids in platelet biogenesis and is a reservoir for hematopoietic stem and progenitor cells (HSPCs). However, lung HECs have never been described. Here we demonstrate that the fetal lung is a novel source of HECs that have the functional capacity to undergo EHT to produce de novo HSPCs and their resultant progeny. Explant cultures of murine and human fetal lungs display adherent endothelial cells transitioning into floating hematopoietic cells accompanied by the gradual loss of an endothelial signature. Flow cytometric and functional assessment of fetal lung explants showed the production of multipotent HSPCs that expressed the EHT and pre-HSPC markers EPCR, CD41, CD43 and CD44. scRNA-Seq and small molecule modulation demonstrated that fetal lung HECs rely on canonical signaling pathways to undergo EHT, including TGFβ/BMP, Notch and YAP. Immunofluorescent stains of fixed frozen fetal lung samples showed discrete regions of clustered hematopoietic cells expressing VE-cadherin, CD41 and CD45 adjacent to fetal lung vasculature. Notably, these clusters were oriented away from the vascular lumen, suggesting they may be fated to remain within the lung interstitium. Collectively, these data strongly support the concept that post-AGM development, functional HECs are present in the fetal lung establishing this location as a potential extramedullary site of de-novo hematopoiesis. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract C032: Combination CAF/myeloid targeting in PDAC

Abstract Immunotherapy has revolutionized clinical care for many cancers, yet these treatments fail to control disease in many patients and new strategies are needed to improve anti-tumor immunity and enhance response rates. There is great need for an increased understanding of the cellular crosstalk within tumors and the identification of stromal and immune populations involved in shaping the tumor microenvironment (TME). Local immunosuppression (LIS) is one of the striking hallmarks of Pancreatic Ductal Adenocarcinoma (PDAC), a disease that is highly resistant to existing immunotherapies. Oncogenic Kras activation in tumor cells promotes the invasion and proliferation of tumor-supporting stromal cells, while excluding cancer-targeted cytotoxic T cells. LIS is mediated by multiple subtypes of cancer-associated fibroblasts (CAFs) and myeloid cells resident within the tumor parenchyma. Multiple prior attempts to reverse LIS in PDAC by targeting individual stromal cell populations have been unsuccessful, alluding to the complexity of stromal crosstalk within the TME. The stromal diversity of PDAC complicates investigating paracrine cascades involving multiple cell types. To decipher diverse drug effects on altering the TME, we employ in vivo studies in mouse models recapitulating the human disease, as well as a novel tumor explant model that enables the short-term culture of intact human or murine PDAC. Importantly, PDAC explants maintain their histopathological architecture and cellular diversity over time. This medium-throughput platform allows for testing of multiple drugs and mechanistic hypotheses in the native PDAC TME. We show in preliminary data that Smoothened inhibition (SMOi) decreases proliferation and activity of myCAFs, but provokes the expansion of CD11b-positive myeloid cells in vivo. Thus, we hypothesize that LIS in PDAC is maintained by a delicate balance between myCAFs and myeloid cells, preventing effective T cell invasion. Single cell RNA-seq data comparing ctrl vs. SMOi-treated murine PDAC elucidates stromal subpopulations involved in the LIS phenotype and guides the identification of myeloid subtypes emerging after SMOi. Strikingly, we demonstrated that simultaneous SMOi and targeting myeloid cells via anti-Gr1 or CCR1 inhibition (CCR1i) significantly elevates cytotoxic T cell numbers within the TME. We are currently investigating whether the activity of these T cells may be further potentiated through combination with immunomodulatory agents. By testing various treatment combination in the same TME, we will identify the best synergistic effects for future immunotherapy approaches in human PDAC. In summary, we are elucidating the complex mechanism behind LIS in PDAC by employing our novel explant culture system alongside in vivo studies. We aim to develop a translatable regimen to neutralize LIS, reactivating the cytotoxic T cells in the tumor periphery to invade, proliferate, and attack cancer cells. Citation Format: Marie C. Hasselluhn, Lukas J. Vlahos, Dafydd Thomas, Alvaro Curiel Garcia, Amanda R. Decker, Tanner C. Dalton, Stephen A. Sastra, Carmine F. Palermo, Andrea Califano, Kenneth P. Olive. Combination CAF/myeloid targeting in PDAC [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 C032.

Abstract B071: Preclinical evaluation of RM-042, an orally bioavailable inhibitor of GTP-RAS, in models of pancreatic ductal adenocarcinoma

Abstract Approximately 95% of pancreatic ductal adenocarcinoma (PDAC) cases are driven by activating mutations in KRAS, accounting for over 45,000 deaths per year in the United States. In KRAS-mutant cells, KRAS protein exists predominantly in an active, GTP-bound state, leading to excessive oncogenic signaling via RAF/MEK/ERK and other effector pathways. After decades of anticipation, the first therapeutic inhibitors of this oncoprotein target a specific mutant allele (KRASG12C) and have shown promising results in this rare subset of human PDAC patients (1-2% of cases). While these encouraging clinical results bolster support for targeting RAS in human PDAC, most patients harbor KRASG12D and KRASG12V mutations and remain unserved by targeted therapies. Here we report preclinical studies of RM-042, a first-in-class, potent, oral, small molecule inhibitor of GTP-bound RAS proteins, designed to treat cancers driven by a variety of RAS mutations. RMC-6236, a clinical candidate and RM-042, a tool compound, both act through non-covalent binding to an abundant intracellular chaperone protein, cyclophilin A (CypA), resulting in a binary complex that engages GTP-bound RAS to form a high-affinity, RAS-selective tri-complex that sterically inhibits RAS binding to effectors. We examined the anti-tumor activity of RM-042 in two complementary preclinical model systems of PDAC. Using primary human PDAC explant cultures (tissue slices cultured intact in optimized media for up to one week), we found that RM-042 potently inhibits phospho-ERK expression (a marker of KRAS/RAF/MEK pathway activity), in a concentration-dependent manner. Loss of pERK expression in tumors was also associated with reduced viability, and altered stromal content. Pilot studies in the K-rasLSL.G12D/+, p53LSL.R172H/+, Pdx1Cretg/+ (KPC) mouse model of PDAC found that RM-042 induced tumor stabilizations and regressions associated with loss of pERK expression, and alterations in apoptosis and proliferation, at tolerated doses. Ongoing studies will determine the duration of tumor responses and assess the mechanisms by which RM-042 may impact both epithelial and stromal cell populations. Together, these studies provide complementary evidence from in vivo murine and ex vivo human models for anti-tumor activity in response to GTP-Ras inhibition, supporting the inclusion of PDAC patients in future clinical trials of RMC-6236. Citation Format: Urszula N. Wasko, Stephen A. Sastra, Carmine F. Palermo, David Wildes, Mallika Singh, Kenneth P. Olive. Preclinical evaluation of RM-042, an orally bioavailable inhibitor of GTP-RAS, in models of pancreatic ductal adenocarcinoma [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 B071.

Conceptus-derived cytokines interleukin-1β and interferon-γ induce the expression of acute phase protein serum amyloid A3 in endometrial epithelia at the time of conceptus implantation in pigs.

Serum amyloid A3 (SAA3), an acute phase response protein, plays important roles in opsonization, antimicrobial activity, chemotactic activity, and immunomodulation, but its expression, regulation, and function at the maternal-conceptus interface in pigs are not fully understood. Therefore, we determined the expression of SAA3 in the endometrium throughout the estrous cycle and at the maternal-conceptus interface during pregnancy. Endometrial tissues from pigs at various stages of the estrous cycle and pregnancy and with conceptuses derived from somatic cell nuclear transfer (SCNT), conceptus tissues during early pregnancy, and chorioallantoic tissues during mid- to late pregnancy were obtained and the expression of SAA3 was analyzed. The effects of the steroid hormones, interleukin-1β (IL1B), and interferon-γ (IFNG) on the expression of SAA3 were determined in endometrial explant cultures. SAA3 was expressed in the endometrium during the estrous cycle and pregnancy, with the highest level on day 12 of pregnancy. The expression of SAA3 in the endometrium was significantly higher on day 12 of pregnancy than during the estrous cycle. Early-stage conceptuses and chorioallantoic tissues during mid to late pregnancy also expressed SAA3. The expression of SAA3 was primarily localized to luminal epithelial cells in the endometrium. In endometrial explant cultures, the expression of SAA3 was induced by increasing doses of IL1B and IFNG. Furthermore, the expression of SAA3 decreased significantly in the endometria of pigs carrying conceptuses derived from SCNT on day 12 of pregnancy. These results suggest that the expression of SAA3 in the endometrium during the implantation period increases in response to conceptus-derived IL1B and IFNG. The failure of those appropriate interactions between the implanting conceptus and the endometrium leads to dysregulation of endometrial SAA3 expression, which could result in pregnancy failure. In addition, SAA3 could be a specific endometrial epithelial marker for conceptus implantation in pigs.

Osteochondral Explant Isolation and Culture Under a Compression and Shear Bioreactor.

Osteochondral explants harvested from different species are valuable preclinical ex vivo models for tissue engineering research. In this chapter, we describe the isolation of osteochondral plugs from bovine stifle joints, followed by defect creation, and plug preparation in a straightforward manner before mechanical loading using a compression and shear bioreactor. The method can be adapted to isolate osteochondral plugs from any animal species and to load explants in any type of bioreactor.

M13, an anthraquinone compound isolated from Morinda officinalis promotes the osteogenic differentiation of MSCs by targeting Wnt/β-catenin signaling

BackgroundMorinda officinalis (MO) is a herb used in Traditional Chinese Medicine (TCM) for the treatment of osteoporosis. M13, a MO-based anthraquinone compound is known to suppress osteoclast activity. However, whether M13 promotes MSCs osteogenic differentiation and its potential mechanism remains unknown. PurposeTo examine the influence of M13 on MSCs proliferation and osteogenic differentiation and elucidate the underlying mechanism. Methods/Study designsThe effect of M13 exposure on MSCs proliferation was assessed via CCK8 assay, clone formation assay, immunofluorescence, RT-qPCR, and Western blot. The M13-mediated osteogenesis in vitro and ex vivo were evaluated via ALP and Alizarin red S staining, osteogenesis-associated gene (Runx2, Col1a1 and Opn) expression, and fetal limb explants culture. Molecular docking was employed for target signal pathway screening. The potential signaling mechanisms of M13-promoted MSCs osteogenic differentiation were analyzed by introducing XAV939 (Wnt/β-catenin signaling inhibitor). ResultsM13 induced certain obvious positive effects on MSCs proliferation and osteogenic differentiation. Treatment with M13 enhanced MSCs viability and clone numbers. Meanwhile, M13 promoted osteogenic gene expression, enhanced ALP intensity and Alizarin red S staining in MSCs. In terms of mechanism, M13 strongly interacted with the docking site of the WNT signaling complex, thereby activating the Wnt/β-catenin pathway. Furthermore, the M13-mediated osteogenic effect was partially inhibited by XAV939 both in vitro and ex vivo, which confirmed that the Wnt/β-catenin axis is a critical regulator of M13-induced osteogenic differentiation of MSCs. ConclusionOur study elucidated for the first time that M13 significantly promoted osteogenic differentiation of MSCs via stimulation of the Wnt/β-catenin pathway in vitro and ex vivo.Our findings offered new additional evidence to support the MO or M13-based therapy of osteoporosis.

Reactive astrocytes transduce inflammation in a blood-brain barrier model through a TNF-STAT3 signaling axis and secretion of alpha 1-antichymotrypsin

Astrocytes are critical components of the neurovascular unit that support blood-brain barrier (BBB) function. Pathological transformation of astrocytes to reactive states can be protective or harmful to BBB function. Here, using a human induced pluripotent stem cell (iPSC)-derived BBB co-culture model, we show that tumor necrosis factor (TNF) transitions astrocytes to an inflammatory reactive state that causes BBB dysfunction through activation of STAT3 and increased expression of SERPINA3, which encodes alpha 1-antichymotrypsin (α1ACT). To contextualize these findings, we correlated astrocytic STAT3 activation to vascular inflammation in postmortem human tissue. Further, in murine brain organotypic cultures, astrocyte-specific silencing of Serpina3n reduced vascular inflammation after TNF challenge. Last, treatment with recombinant Serpina3n in both ex vivo explant cultures and in vivo was sufficient to induce BBB dysfunction-related molecular changes. Overall, our results define the TNF-STAT3-α1ACT signaling axis as a driver of an inflammatory reactive astrocyte signature that contributes to BBB dysfunction.

Ependyma: a new target for autoantibodies in neuromyelitis optica?

Neuromyelitis optica (NMO) is an autoimmune demyelinating disease of the central nervous system characterized by the presence of autoantibodies (called NMO-IgG) targeting aquaporin-4. Aquaporin-4 is expressed at the perivascular foot processes of astrocytes, in the glia limitans, but also at the ependyma. Most studies have focused on studying the pathogenicity of NMO-IgG on astrocytes, and NMO is now considered an astrocytopathy. However, periependymal lesions are observed in NMO suggesting that ependymal cells could also be targeted by NMO-IgG. Ependymal cells regulate CSF-parenchyma molecular exchanges and CSF flow, and are a niche for sub-ventricular neural stem cells. Our aim was to examine the effect of antibodies from NMO patients on ependymal cells. We exposed two models, i.e. primary cultures of rat ependymal cells and explant cultures of rat lateral ventricular wall whole mounts, to purified IgG of NMO patients (NMO-IgG) for 24 hours. We then evaluated the treatment effect using immunolabelling, functional assays, ependymal flow analysis and bulk RNA sequencing. For each experiment, the effects were compared with those of purified IgG from a healthy donors and non-treated cells. We found that: (i) NMO-IgG induced aquaporin-4 agglomeration at the surface of ependymal cells and induced cell enlargement in comparison to controls. In parallel, it induced an increase in gap junction connexin-43 plaque size; (ii) NMO-IgG altered the orientation of ciliary basal bodies and functionally impaired cilia motility; (iii) NMO-IgG activated the proliferation of sub-ventricular neural stem cells; (iv) treatment with NMO-IgG up-regulated the expression of pro-inflammatory cytokines and chemokines in the transcriptomic analysis. Our study showed that NMO-IgG can trigger an early and specific reactive phenotype in ependymal cells, with functional alterations of intercellular communication and cilia, activation of sub-ventricular stem cell proliferation and the secretion of pro-inflammatory cytokines. These findings suggest a key role for ependymal cells in the early phase of NMO lesion formation.

Xenopus Explants and Transplants.

There is a long tradition of testing the developmental potential and competency of different regions of the embryo by transplanting tissue to novel locations or by growing isolated tissue in explant culture. The protocols introduced here describe several types of embryological manipulations in Xenopus that are particularly useful for analyzing tissue inductive signals, cell migration, and organogenesis. These techniques draw upon classical approaches that are quite easy to perform in both Xenopus laevis and Xenopus tropicalis embryos.

Elicitor and precursor-induced approaches to enhance the in vitro production of L-DOPA from cell cultures of Mucuna pruriens

Elicitation and precursor feeding are two important strategies in the in vitro techniques to enhance metabolite production to meet the demand of mankind. The secondary metabolites produced by the plants are extensively used in pharmaceutical, food and agro-chemical industries. One such metabolite is 3,4 dihydroxyphenylalanine ( L -DOPA) produced from Mucuna pruriens (L.) DC. is used since ancient times to treat Parkinson’s disease. Though all parts produce L -DOPA, the seed has the highest quantity. To overcome the extensive usage of the natural sources whose growth and metabolite production is highly dependent on edaphic and ecological factors, in vitro techniques like establishing cell culture for continuous production of metabolites, precursor feeding and elicitation of cell cultures to enhance the metabolite production has been reported in the present study. Callus was developed from the in vitro leaf explant and cell suspension culture was established in the liquid Murashige and Skoog’s medium fortified with 0.5 mg/L picloram . Amino acid precursors like tyrosine , phenylalanine and chemical elicitors like methyl jasmonate , salicylic acid, sodium nitroprusside and silver nitrate were exposed to cell cultures for different periods (3, 6 and 9 days respectively). The precursors showed a better response in enhancing both the biomass and L -DOPA when compared to the elicitors. 500 and 1000 mg/L tyrosine showed a 1.6- and an 8.1-fold increase in biomass and L -DOPA production respectively when supplemented with MS media . However, though all the elicitors enhanced the L -DOPA production by 1.1–3.3-folds they did not show much significant increase in biomass. Precursor feeding approaches enhanced the metabolite considerably more than the elicitor treatment. Based on the productivity (Biomass × L -DOPA conc.) precursors like Tyrosine>Phenylalanine and elicitors like Sodium nitroprusside>Silver nitrate>Methyl jasmonate>Salicylic acid showed better response. • First report to elicit L -DOPA from Mucuna pruriens cell cultures using Phenylalanine. • A highest of 8.1-fold increase of L -DOPA was achieved using Tyrosine as a precursor. • Among the elicitors, a 3.3-fold increase of L -DOPA was seen using sodium nitroprusside. • On the basis of productivity, Tyrosine and sodium nitroprusside showed best results.

OR16-5 Riluzole suppresses growth and enhances response to endocrine therapy in ER+ breast cancer

Abstract Approximately 75% of breast cancers are classified as hormone receptor-positive, most of which are estrogen receptor alpha-positive (ER+), which is the primary driver of growth in these tumors. Consequently, endocrine or anti-estrogen therapy is used for treatment. However, despite the benefits of endocrine therapeutics, treatment-resistant relapse is a significant problem that affects 30-50% of patients who initially respond to endocrine therapy. Therefore, studying resistance to endocrine therapy is critical for breast cancer research.We have shown that invasive lobular breast cancer (ILC) cells resistant to the anti-estrogen tamoxifen increase their expression of metabotropic glutamate receptors (GRMs/mGluRs). GRMs are well-known to play important roles in learning and memory in the brain and protect neurons from excitotoxicity (cell death caused by prolonged exposure to neurotransmitters). It is now appreciated that this pro-survival function of GRMs can be highjacked by cancer cells, including breast cancer. Thus, targeting GRM signaling could prove a valuable therapeutic strategy. We chose to target the GRM signaling pathway with the FDA-approved drug Riluzole currently being used to treat amyotrophic lateral sclerosis.In this study, we test the ability of Riluzole to reduce cell growth, alone and in combination with endocrine therapy, in a diverse set of ER+ invasive ductal and lobular breast cancer-derived cell lines, primary breast tumor explant cultures, and the estrogen-independent, ESR1-mutated, ILC-derived patient-derived xenograft model HCI-013EI. In addition to measuring tumor growth rate and size, primary tumors and organs were collected for immunohistochemistry analysis.Riluzole as a single agent suppressed the growth of ER+ invasive ductal and lobular breast cancer cell lines in vitro, inducing differential histologic subtype-associated cell cycle arrest (G0-G1 for ductal, G2-M for lobular). In tamoxifen-resistant ILC cells, Riluzole induced apoptotic and ferroptotic cell death, and inhibited phosphorylation of focal adhesion kinase. Riluzole combined with either fulvestrant or 4-hydroxytamoxifen additively or synergistically suppressed ER+ breast cancer cell growth in vitro. Using proliferating cell nuclear antigen (PCNA) staining as a proxy for cell proliferation, the combination of Riluzole plus Fulvestrant significantly reduced PCNA in a patient-derived explant model (t-test p = 0.013). The in vivo experiment showed reduced tumor size and growth between the control and combination treatments. However, unlike in the in vitro experiment, there was little difference between the single-agent fulvestrant and the combination groups. The observed difference between the in vitro and in vivo study may be attributed to the bioavailability of Riluzole in mice.In conclusion, our results show that Riluzole enhances response to endocrine therapy in ER+ breast cancer. Presentation: Sunday, June 12, 2022 12:00 p.m. - 12:15 p.m.

In vivo ligamentogenesis in embroidered poly(lactic-co-ε-caprolactone) / polylactic acid scaffolds functionalized by fluorination and hexamethylene diisocyanate cross-linked collagen foams

Although autografts represent the gold standard for anterior cruciate ligament (ACL) reconstruction, tissue-engineered ACLs provide a prospect to minimize donor site morbidity and limited graft availability. This study characterizes the ligamentogenesis in embroidered poly(L-lactide-co-ε-caprolactone) (P(LA-CL)) / polylactic acid (PLA) constructs using a dynamic nude mice xenograft model. (P(LA-CL))/PLA scaffolds remained either untreated (co) or were functionalized by gas fluorination (F), collagen foam cross-linked with hexamethylene diisocyanate (HMDI) (coll), or F combined with the foam (F + coll). Cell-free constructs or those seeded for 1 week with lapine ACL ligamentocytes were implanted into nude mice for 12 weeks. Following explantation, cell vitality and content, histo(patho)logy of scaffolds (including organs: liver, kidney, spleen), sulphated glycosaminoglycan (sGAG) contents and biomechanical properties were assessed.Scaffolds did not affect mice weight development and organs, indicating no organ toxicity. Moreover, scaffolds maintained their size and shape and reflected a high cell viability prior to and following implantation. Coll or F + coll scaffolds seeded with cells yielded superior macroscopic properties compared to the controls. Mild signs of inflammation (foreign-body giant cells and hyperemia) were limited to scaffolds without collagen. Microscopical score values and sGAG content did not differ significantly. Although remaining stable after explantation, elastic modulus, maximum force, tensile strength and strain at Fmax were significantly lower in explanted scaffolds compared to those before implantation, with no significant differences between scaffold subtypes, except for a higher maximum force in F + coll compared with F samples (in vivo). Scaffold functionalization with fluorinated collagen foam provides a promising approach for ACL tissue engineering.Graphical abstracta Lapine anterior cruciate ligament (LACL): red arrow, posterior cruciate ligament: yellow arrow. Medial anterior meniscotibial ligament: black arrow. b Explant culture to isolate LACL fibroblasts. c Scaffold variants: co: controls; F: functionalization by gas-phase fluorination; coll: collagen foam cross-linked with hexamethylene diisocyanate (HMDI). c1-2 Embroidery pattern of the scaffolds. d Scaffolds were seeded with LACL fibroblasts using a dynamical culturing approach as depicted. e Scaffolds were implanted subnuchally into nude mice, fixed at the nuchal ligament and sacrospinal muscle tendons. f Two weeks after implantation. g Summary of analyses performed. Scale bars 1 cm (b, d), 0.5 cm (c). (sketches drawn by G.S.-T. using Krita 4.1.7 [Krita foundation, The Netherlands]).