Hepatocyte Growth Factor Secretion Research Articles

Cryopreserved cGMP-compliant human pluripotent stem cell-derived hepatic progenitors rescue mice from acute liver failure through rapid paracrine effects on liver cells

BackgroundLiver transplantation remains the only curative treatment for end-stage liver diseases. Unfortunately, there is a drastic organ donor shortage. Hepatocyte transplantation emerged as a viable alternative to liver transplantation. Considering their unique expansion capabilities and their potency to be driven toward a chosen cell fate, pluripotent stem cells are extensively studied as an unlimited cell source of hepatocytes for cell therapy. It has been previously shown that freshly prepared hepatocyte-like cells can cure mice from acute and chronic liver failure and restore liver function.MethodsHuman PSC-derived immature hepatic progenitors (GStemHep) were generated using a new protocol with current good manufacturing practice compliant conditions from PSC amplification and hepatic differentiation to cell cryopreservation. The therapeutic potential of these cryopreserved cells was assessed in two clinically relevant models of acute liver failure, and the mode of action was studied by several analytical methods, including unbiased proteomic analyses.ResultsGStemHep cells present an immature hepatic phenotype (alpha-fetoprotein positive, albumin negative), secrete hepatocyte growth factor and do not express major histocompatibility complex. A single dose of thawed GStemHep rescue mice from sudden death caused by acetaminophen and thioacetamide-induced acute liver failure, both in immunodeficient and immunocompetent animals in the absence of immunosuppression. Therapeutic biological effects were observed as soon as 3 h post-cell transplantation with a reduction in serum transaminases and in liver necrosis. The swiftness of the therapeutic effect suggests a paracrine mechanism of action of GStemHep leading to a rapid reduction of inflammation as well as a rapid cytoprotective effect with as a result a proteome reprograming of the host hepatocytes. The mode of action of GStemHep relie on the alleviation of inhibitory factors of liver regeneration, an increase in proliferation-promoting factors and a decrease in liver inflammation.ConclusionsWe generated cryopreserved and current good manufacturing practice-compliant human pluripotent stem cell-derived immature hepatic progenitors that were highly effective in treating acute liver failure through rapid paracrine effects reprogramming endogenous hepatocytes. This is also the first report highlighting that human allogeneic cells could be used as cryopreserved cells and in the absence of immunosuppression for human PSC-based regenerative medicine for acute liver failure.

Function of mast cell and bile-cholangiocarcinoma interplay in cholangiocarcinoma microenvironment

ObjectiveThe correlation between cholangiocarcinoma (CCA) progression and bile is rarely studied. Here, we aimed to identify differential metabolites in benign and malignant bile ducts and elucidate the generation, function and...

Therapeutic potential of exosome derived from hepatocyte growth factor-overexpressing adipose mesenchymal stem cells in TGFβ1-stimulated hepatic stellate cells.

Cirrhosis is a familiar end-stage of multiple chronic liver diseases. The gene-modified mesenchymal stem cells (MSCs) have become one of the most promising schemes for the treatment of cirrhosis. MSCs exhibit their therapeutic role mainly by secreting hepatocyte growth factor (HGF). The aim of this research was to probe the anti-fibrosis role of exosomes secreted by HGF modified-mouse adipose MSCs (ADMSCs) on activated hepatic stellate cells (HSCs) and to preliminarily explore the possible mechanism. Firstly, mouse ADMSCs were isolated and identified. Quantitative real-time polymerase chain reaction verified the transfection efficiency of ADMSC transfected with HGF lentivirus. Exosomes derived from ADMSC transfecting negative control/HGF (ADMSCNC-Exo/ADMSCHGF-Exo) were extracted by density gradient centrifugation. HSCs were allocated to the control, TGF-β, TGF-β + ADMSC-Exo, TGF-β + ADMSCNC-Exo, and TGF-β + ADMSCHGF-Exo groups. Moreover, all mice were distributed to the control, CCl4 (40% CCl4 in olive oil), CCl4+ADMSC-Exo, CCl4+ADMSCNC-Exo, and CCl4+ADMSCHGF-Exo groups. Exosomes derived from ADMSCs with or without HGF transfection suppressed HSC activation, as evidenced by attenuating cell viability and cell cycle arrest at S phase but inducing apoptosis. Moreover, ADMSC-Exo, ADMSCNC-Exo, and ADMSCHGF-Exo effectively repressed the gene and protein levels of α-SMA, Col-I, Rho A, Cdc42, and Rac1 in TGF-β-treated HSCs, and ADMSCHGF-Exo had the best effect. ADMSCHGF-Exo had a stronger regulatory effect on serum liver index than ADMSCNC-Exo in CCl4-induced mice. In conclusion, ADMSCHGF-Exo alleviated liver fibrosis by weakening the Rho pathway, thus reducing collagen production.

Human corneal epithelial cell and fibroblast migration and growth factor secretion after rose bengal photodynamic therapy (RB-PDT) and the effect of conditioned medium.

To investigate human corneal epithelial cell and fibroblast migration and growth factor secretion after rose bengal photodynamic therapy (RB-PDT) and the effect of conditioned medium (CM). A human corneal epithelial cell line (HCE-T), human corneal fibroblasts (HCF) and keratoconus fibroblasts (KC-HCF) have been used. Twenty-four hours after RB-PDT (0.001% RB concentration, 565 nm wavelength illumination, 0.17 J/cm2 fluence) cell migration rate using scratch assay and growth factor concentrations in the cell culture supernatant using ELISA have been determined. In addition, the effect of CM has been observed. RB-PDT significantly reduced migration rate in all cell types, compared to controls (p≤0.02). Migration rate of HCE-T cultures without RB-PDT (untreated) was significantly higher using HCF CM after RB-PDT, than using HCF CM without RB-PDT (p<0.01). Similarly, untreated HCF displayed a significantly increased migration rate with HCE-T CM after RB-PDT, compared to HCE-T CM without treatment (p<0.01). Furthermore, illumination alone and RB-PDT significantly decreased keratinocyte growth factor (KGF) concentration in HCF and KC-HCF supernatant, and RB-PDT significantly decreased soluble N-Cadherin (SN-Cad) concentration in HCF supernatant, compared to controls (p<0.01 for all). In HCE-T CM, RB-PDT increased hepatocyte growth factor (HGF) and basic fibroblast growth factor (FGFb) concentration (p≤0.02), while decreasing transforming growth factor β (TGF-β) concentration (p<0.01). FGFb concentration increased (p<0.0001) and TGF-β concentration decreased (p<0.0001) in HCF CM, by RB-PDT. Epidermal growth factor (EGF), HGF, and TGF-β concentration decreased (p≤0.03) and FGFb concentration increased (p<0.01) in KC-HCF CM, using RB-PDT. HCE-T, HCF and KC-HCF migration rate is reduced 24 hours after RB-PDT. In contrast, HCE-T migration is enhanced using HCF CM after RB-PDT, and HCF migration rate is increased through HCE-T CM following RB-PDT. Modulation of EGF, KGF, HGF, FGFb, TGF-β and N-Cadherin secretion through RB-PDT may play an important role in corneal wound healing.

Cross-talk between gastric cancer and hepatic stellate cells promotes invadopodia formation during liver metastasis.

In gastric cancer (GC), the liver is a common organ for distant metastasis, and patients with gastric cancer with liver metastasis (GCLM) generally have poor prognosis. The mechanism of GCLM is unclear. Invadopodia are special membrane protrusions formed by tumor cells that can degrade the basement membrane and ECM. Herein, we investigated the role of invadopodia in GCLM. We found that the levels of invadopodia-associated proteins were significantly higher in liver metastasis than in the primary tumors of patients with GCLM. Furthermore, GC cells could activate hepatic stellate cells (HSCs) within the tumor microenvironment of liver metastases through the secretion of platelet-derived growth factor subunit B (PDGFB). Activated HSCs secreted hepatocyte growth factor (HGF), which activated the MET proto-oncogene, MET receptor of GC cells, thereby promoting invadopodia formation through the PI3K/AKT pathway and subsequently enhancing the invasion and metastasis of GC cells. Therefore, cross-talk between GC cells and HSCs by PDGFB/platelet derived growth factor receptor beta (PDGFRβ) and the HGF/MET axis might represent potential therapeutic targets to treat GCLM.

HGF Regulation in the Tumor-Supportive Microenvironment Combined with Pan-PIM Kinases Inhibition in Malignant Cells As a Multi-Target Approach for Acute Myeloid Leukemia

Background: In acute myeloid leukemia (AML), persistent clonal proliferation of malignant myeloid cells results in high severity and low cure potential. Resistance to currently available chemotherapeutics and disease relapse remains an important concern worldwide. Several molecular targets play key roles in impairing therapeutic response, including PIM-kinases, a serine/threonine kinase family associated with the progression of hepatocyte growth factor (HGF)-driven cancers through its receptor, MET. We reported that enhanced HGF secretion by bone marrow (BM) mesenchymal stromal cells (MSC) correlated with low levels of the serine protease inhibitor kunitz-type2 (SPINT2) in AML (Roversi et al., 2019), which are affected by immunosuppressive properties of the tumor microenvironment (TME). However, the interplay between SPINT2, HGF, and PIM-kinases in the TME is yet to be elucidated and could represent an important multi-target therapeutic strategy in hematological cancers. Aims: Given the importance of HGF to AML prognosis and progression, we analyzed the interplay between SPINT2, HGF, and pan-PIM kinases by evaluating the gene expression and the effects of a novel pan-PIM kinase inhibitor, PIM447 (Novartis), in leukemia cells and in the TME. Methods: SPINT2, HGF, PIM1, PIM2 and PIM3 mRNA expression data were obtained from OHSU (2018) AML study and TCGA (cBio Portal). Correlation analysis was obtained by Spearman's coefficient (r). A total of 5 healthy donors (HD) and 5 de novo AML patients BMMSC (CD90+CD105+CD73+CD45-CD34-CD31-HLA-DR-) were treated with 5-Azacitidine (Aza), and evaluated for SPINT2 gene expressions. Myeloid (OCI-AML3, MOLM13, U937) and lymphoblastic (RS4;11, Jurkat) leukemia cells were treated with increasing concentrations of PIM447, and the 101nd apoptosis rates were determined. Peripheral blood and BM mononuclear cells (PBMC/BMMC) from HD were tested for cytotoxicity. Leukemia cells were also examined for reactive oxygen species (ROS), nitric oxide (NO) production, and mitochondrial membrane potential (ΔΨm). To verify PIM447 action onto an immunosuppressive TME, cocultures of leukemia cells and monocytes or monocyte-derived macrophages (MDMs) were performed. To understand the interplay between SPINT2 and PIM-kinases, cocultures of SPINT2-overexpressed MSC and leukemia cells, were performed. Statistical analyzes were performed using Kruskal Wallis, ANOVA or Mann-Whitney tests ( P&amp;lt;.05). Results: The analysis of gene expression performed in MC from 188 AML patients (age median: 57.5 years [range: 18-88]) showed that levels of HGF correlated positively with PIM1, PIM2, and PIM3 ((r) =.8727;.9094;.9093, respectively, P&amp;lt;.0001) and negatively with SPINT2 (r=-.06402). We also verified that SPINT2 mRNA expression was upregulated in AML-BMMSC ( P&amp;lt;.05) after Aza treatment when compared to HD-BMMSC, suggesting that this gene is methylated in AML. Our in vitro results showed PIM447 treatment (2-20μM) yielded reduced cell viability and increased apoptosis in leukemia cells [IC50 range: 12.2-22.2μM, P&amp;lt;.01] without affecting HD cells. PIM447 reduced ROS, NO production, and ΔΨm (all P&amp;lt;.0001) in all cell lines. PIM447 treatment of macrophages differentiated from HD-PBMC revealed a shorter population of M2 macrophages (HLA-DR-CD206+) ( P&amp;lt;.001) and an increased population of M1 macrophages (HLA-DR+CD80+) ( P&amp;lt;.05). Moreover, PIM447 promoted apoptosis of leukemia cells co-cultured with MDMs ( P&amp;lt;.0001). Interestingly, co-culture analyses of SPINT2-overexpressed MSC enhanced PIM447 cytotoxic activity towards leukemia cells (P&amp;lt;.05) and reduced cell-cell adhesion by diminishing CD49b, CD49d, and CD49e (P&amp;lt;.05), suggesting an interplay between SPINT2 and pan-PIM kinases Conclusion: Collectively, our data identify a novel pan-PIM kinase inhibitor, PIM447, which effectively induces leukemia cell apoptosis through reducing MET receptor activation as well as acts on their tumor-supportive microenvironment by inducing a phenotypic shift from immunosuppressive M2 to anti-tumor M1 macrophages. PIM447 also interplays with SPINT2/HGF pathway in the TME, reducing AML progression. These findings suggest that a pivotal strategy to treat AML could be a multi-target therapy, acting both on leukemia cells and in the tumor microenvironment. Funding: FAPESP #2017/21801-2, #2019/25247-5, #2021/05320-0; CNPq #303405/2018-0.

Increased Hepatocyte Growth Factor Secretion by Placenta-Derived Mesenchymal Stem Cells Improves Ovarian Function in an Ovariectomized Rat Model via Vascular Remodeling by Wnt Signaling Activation.

The vascular network contributes to the development of follicles. However, the therapeutic mechanism between vascular remodeling and ovarian functions is still unclear. Therefore, we demonstrated whether increased HGF by placenta-derived mesenchymal stem cells (PD-MSCs) improves ovarian function in an ovariectomized rat model via vascular remodeling by Wnt signaling activation. We established a half-ovariectomized rat model in which damaged ovaries were induced by ovariectomy of half of each ovary, and PD-MSCs (5 × 105 cells) were transplanted by intravenous injection. Three weeks after transplantation, rats in all groups were sacrificed. We examined the secretion of HGF by PD-MSCs through culture medium. The vascular structure in injured ovarian tissues was restored to a greater extent in the PD-MSC transplantation (Tx) group than in the nontransplantation (NTx) group (* p < 0.05). The expression of genes related to Wnt signaling (e.g., LRP6, GSK3β, β-catenin) was significantly increased in the Tx group compared to the NTx group (* p < 0.05). However, the expression of genes related to vascular permeability (e.g., Asef, ERG3) was significantly decreased in the Tx group compared to the NTx group (* p < 0.05). Follicular development was improved in the Tx group compared to the NTx group (* p < 0.05). Furthermore, to evaluate vascular function, we cocultivated PD-MSCs after human umbilical vein endothelial cells (HUVECs) with lipopolysaccharide (LPS), and we analyzed the vascular formation assay and dextran assay in HUVECs. Cocultivation of PD-MSCs with injured HUVECs enhanced vascular formation and decreased endothelial cell permeability (* p < 0.05). Also, cocultivation of PD-MSCs with explanted ovarian tissues improved follicular maturation compared to cocultivation of the Wnt inhibitor-treated PD-MSCs with explanted ovarian tissues. Therefore, HGF secreted by PD-MSCs improved ovarian function in rats with ovarian dysfunction by decreasing vascular permeability via Wnt signaling.

Bone marrow-derived mesenchymal stem cells reactive oxygen species-responsively secreting hepatocyte growth factor for effective treatment of ischemia-reperfusion injury in liver transplantation

Bone marrow-derived mesenchymal stem cells reactive oxygen species-responsively secreting hepatocyte growth factor for effective treatment of ischemia-reperfusion injury in liver transplantation

Mesenchymal stem cell-derived HGF attenuates radiation-induced senescence in salivary glands via compensatory proliferation

Background & AimIrradiation of the salivary glands during head and neck cancer treatment induces cellular senescence in response to DNA damage and contributes to radiation-induced hyposalivation by affecting the salivary gland stem/progenitor cell (SGSC) niche. Cellular senescence, such as that induced by radiation, is a state of cell-cycle arrest, accompanied by an altered pro-inflammatory secretome known as the senescence-associated secretory phenotype (SASP) with potential detrimental effects on the surrounding microenvironment. We hypothesized that the pro-regenerative properties of mesenchymal stem cells (MSCs) may attenuate cellular senescence post-irradiation. Therefore, here we evaluated the effects of adipose-derived MSCs (ADSCs) on the radiation-induced response of salivary gland organoids (SGOs). MethodsProteomic analyses to identify soluble mediators released by ADSCs co-cultured with SGOS revealed secretion of hepatocyte growth factor (HGF) in ADSCs, suggesting a possible role in the stem cell crosstalk. Next, the effect of recombinant HGF in the culture media of ex vivo grown salivary gland cells was tested in 2D monolayers and 3D organoid models. ResultsTreatment with HGF robustly increased salivary gland cell proliferation. Importantly, HGF supplementation post-irradiation enhanced proliferation at lower doses of radiation (0, 3, 7 Gy), but not at higher doses (10, 14 Gy) where most cells stained positive for senescence-associated beta-galactosidase. Furthermore, HGF had no effect on the senescence-associated secretory phenotype (SASP) of irradiated SGOs, suggesting there may be compensatory proliferation by cell-division competent cells instead of a reversal of cellular senescence after irradiation. ConclusionADSCs may positively influence radiation recovery through HGF secretion and can promote the ex vivo expansion of salivary gland stem/progenitor cells to enhance the effects of co-transplanted SGSC.

Poly(ethylene glycol)-Based Hydrogel Microcarriers Alter Secretory Activity of Genetically Modified Mesenchymal Stromal Cells.

In order to scale up culture therapeutic cells, such as mesenchymal stromal cells (MSCs), culture in suspension bioreactors using microcarriers (μCs) is preferred. However, the impact of microcarrier type on the resulting MSC secretory activity has not been investigated. In this study, two poly(ethylene glycol) hydrogel formulations with different swelling ratios (named "stiffer" and "softer") were fabricated as μC substrates to culture MSCs and MSCs genetically modified to express the interleukin-1 receptor antagonist (IL-1Ra-MSCs). Changes in cell number, secretory and angiogenic activity, and changes in MAPK signaling were evaluated when cultured on hydrogel μCs, as well as on tissue culture plastic-based Synthemax μCs. We demonstrated that culture on stiffer μCs increased secretion of IL-1Ra compared to culture on Synthemax μCs by IL-1Ra-MSCs by 1.2- to 1.6-fold, as well as their in vitro angiogenic activity, compared to culture on Synthemax μCs, while culture on both stiffer and softer μCs altered the secretion of several other factors compared to culture on Synthemax μCs. Changes in angiogenic activity corresponded with increased gene expression and secretion of hepatocyte growth factor by MSCs cultured on softer μCs by 2.5- to 6-fold compared to MSCs cultured on Synthemax μCs. Quantification of phosphoprotein signaling with the MAPK pathway revealed broad reduction of pathway activation by IL-1Ra-MSCs cultured on both stiffer and softer μCs compared to Synthemax, where phosphorylated c-Jun, ATF2, and MEK1 were reduced specifically on softer μCs. Overall, this study showed that μC surfaces can influence the secretory activity of genetically modified MSCs and identified associated changes in MAPK pathway signaling, which is a known central regulator of cytokine secretion.

Telocytes protect against lung tissue fibrosis through hexokinase 2-dependent pathway by secreting hepatocyte growth factor.

Pulmonary fibrosis (PF) is one of the common manifestations of end-stage lung disease. Chronic lung failure after lung transplantation is mainly caused by bronchiolitis obliterans syndrome (BOS) and is mainly characterized by lung tissue fibrosis. Pulmonary epithelial-mesenchymal transformation (EMT) is crucial for pulmonary fibrosis. Telocytes (TCs), a new type of mesenchymal cells, play a protective role in various acute injuries. For exploring the anti-pulmonary fibrosis effect of TCs in the BOS model in vitro and the related mechanism, rat tracheal epithelial (RTE) cells were treated with transforming growth factor-β (TGF-β) to simulate lung tissue fibrosis in vitro. The RTE cells were then co-cultured with TCs primarily extracted from rat lung tissue. Western blot, Seahorse XF Analysers and enzyme-linked immunosorbent assay were used to detect the level of EMT and aerobic respiration of RTE cells. Furthermore, anti-hepatocyte growth factor (anti-HGF) antibody was exogenously added to the cultured cells to explore further mechanisms. Moreover, hexokinase 2 (HK2) in RTE cells was knocked down to assess whether it influences the blocking effect of the anti-HGF antibody. TGF-β could induce lung tissue fibrosis in RTE cells in vitro. Nevertheless, TCs co-culture decreased the level of EMT, glucose metabolic indicators (lactate and ATP) and oxygen levels. Furthermore, TCs released hepatocyte growth factor (HGF). Therefore, the exogenous addition of anti-HGF antibody in the co-culture system blocked the anti-lung tissue fibrosis effect. However, HK2 knockdown attenuated the blocking effect of the anti-HGF antibody. In conclusion, TCs can protect against lung tissue fibrosis by releasing HGF, a process dependent on HK2.

Hipoxia modulates the secretion of growth factors of human umbilical cord-derived mesenchymal stem cells.

Mesenchymal stem cell (MSC) has great potential as therapies due its ability to regenerate tissue damage and promote tissue homeostasis. Preconditioning of MSC in low oxygen concentration has been shown to affect the therapeutic potential of these cells. This study aimed to compare the characteristic and secretion of trophic factors of MSCs cultured under hypoxia and normoxia. MSCs were isolated from Wharton's jelly of human umbilical cord (UC) tissue by explant method and characterized by flow cytometry. Following 24 h of CoCl2-induced hypoxic culture, the viability and metabolic activity of MSC were analyzed by trypan blue exclusion test and methyl thiazolyl tetrazolium (MTT) assay, respectively. The secretion of hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF) was assessed in conditioned medium using enzyme-linked immunosorbent assay (ELISA) method. Flow cytometry analysis showed >99% of the population of MSCs cells were positive for CD73 and CD90 and > 62% were positive for CD105. While the cell viability of MSC was not affected by hypoxic cultured condition, the metabolic activity rate of these cells was decreased under hypoxic conditioning. In line with reduced metabolic activity, hypoxic human UC-derived MSC produced less HGF than normoxic counterpart. Compared to normoxic MSC, hypoxic preconditioned MSC secreted higher level of VEGF in the conditioned medium (p < 0.05). Hypoxia decreased the metabolic activity of MSCs associated with the modulation of HGF and VEGF secretions. It is suggested that hypoxia may also affect the therapeutic capacity of MSC cells.

Chemotherapy-induced PDL-1 expression in cancer-associated fibroblasts promotes chemoresistance in NSCLC

ObjectivesA cure for cancer is out of reach for most patients due to chemoresistance. Cancer-associated fibroblasts (CAFs) play a vital role in cancer chemoresistance, but detailed understanding of the process particularly in chemoresistant lung cancer is lacking. In this study, we investigated programmed death-ligand 1 (PDL-1) as a potential biomarker for CAF-induced chemoresistance and evaluated its role and the underlying mechanisms of chemoresistance in non-small cell lung cancer (NSCLC). Materials and MethodsA systemic search of gene expression profiles of multiple tissues in NSCLC was carried out to determine the expression intensities of traditional fibroblast biomarkers and CAF-secreted protumorigenic cytokines. PDL-1 expression in CAFs was analyzed by ELISA, Western blotting, and flow cytometry. Human cytokine array was used to identify specific cytokines secreted from CAFs. Role of PDL-1 in NSCLC chemoresistance was assessed using CRISPR/Cas9 knockdown and various functional assays including MTT, cell invasion, sphere formation, and cell apoptosis. In vivo experiments were conducted using a co-implantation xenograft mouse model with live cell imaging and immunohistochemistry. ResultsWe demonstrated that chemotherapy-stimulated CAFs promoted tumorigenic and stem cell-like properties of NSCLC cells, which contribute to their chemoresistance. Subsequently, we revealed that PDL-1 expression is upregulated in chemotherapy-treated CAFs and is associated with poor prognosis. Silencing PDL-1 expression suppressed CAFs’ ability to promote stem cell-like properties and invasiveness of lung cancer cells, favoring chemoresistance. Mechanistically, an upregulation of PDL-1 in chemotherapy-treated CAFs led to an increase in hepatocyte growth factor (HGF) secretion, which stimulates cancer progression, cell invasion, and stemness of lung cancer cells, while inhibiting apoptosis. ConclusionOur results show that PDL-1-positive CAFs modulate stem cell-like properties of NSCLC cells by secreting elevated HGF, thereby promoting chemoresistance. Our finding supports PDL-1 in CAFs as a chemotherapy response biomarker and as a drug delivery and therapeutic target for chemoresistant NSCLC.

Abstract 552: Stromal facilitated multifactorial resistance to tumor cells against targeted therapies in ALK+ NSCLC

Abstract Despite inducing strong and durable remissions, inhibitors of mutant ALK (ALKi) are not curative for advanced ALK+ lung cancers, as residual tumors eventually develop resistance and relapse. Apart from tumor cells’ intrinsic ability to escape from therapy, there is a growing body of evidence suggesting the contribution of extrinsic factors, produced by cancer-associated fibroblasts (CAFs). Despite the multitude of studies that demonstrated the ability of multiple CAF-produced factors to reduce the sensitivity of tumor cells to ALKi, the contribution of these effects toward responses in vivo remains unresolved. To study the impact of stroma on the ability of tumor cells to survive and develop resistance to ALKi, we derived multiple isolates of fibroblasts from clinical samples. Consistent with previous reports, we found that co-culture with CAFs or CAF-conditioned media protects tumor cells against ALKi. We observed that the degree of protection varies between different CAF isolates. This variability in the extent of protection could be attributed to variability in the levels of secreted hepatocyte growth factor (HGF) a known paracrine mediator of environmental resistance. Moreover, exogenous HGF phenocopied the effect of CAFs while blocking HGF-cMET signaling with neutralizing antibodies or pharmacologically abrogated the protective effect. To test the relevance of these findings in vivo, we took advantage of the inability of murine HGF to activate human cMET by comparing the response of ALK+ xenograft tumors to front-line ALKi alectinib between NSG mice and NSG-derivative strain with humanized HGF. In contrast to the in vitro data, HGF status had only a minimal impact on the remission-relapse dynamics of xenograft tumors. This lack of differences reflected a strong HGF-independent sheltering effect of the stromal niche. Our histological analyses of samples at different points of remission-relapse response revealed that while alectinib potently suppressed tumor cell proliferation, proximity to stroma reduced this cytostatic effect, without impacting cell proliferation in the absence of therapy. To gain insights into the mechanistic underpinning of this HGF-independent effect, we used spatial transcriptomics, comparing stroma-proximal and stroma-distant tumor regions. These analyses, as well as functional validation studies, indicate that stroma-sheltering effects are mediated by multiple mechanisms, acting in an additive fashion. In summary, our studies indicate that therapy resistance of tumor cells reflects the combined action of both intrinsic and microenvironmental factors. Our findings indicate that focusing on a single resistance mechanism at a time is unlikely to induce strong, durable responses. Instead, tackling the issue of therapy resistance necessitates the consideration of multiple resistance mechanisms as well as the moving target nature of tumors under therapy. Citation Format: Bina Desai, Tatiana Miti, Daria Miroshnychenko, Viktoriya Marusyk, Chandler Gatenbee, Menkara Henry, Uwe Rix, Alexander Anderson, Eric Haura, David Basanta, Andriy Marusyk. Stromal facilitated multifactorial resistance to tumor cells against targeted therapies in ALK+ NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 552.

Comprehensive Analysis of Local Anesthetics Affecting Adipose Stem Cells In Vitro.

Adipose stem cells (ASCs) hold a great regenerative capacity because of their differentiation capability and their secretory activity. Thus, ASC survival is of great significance during perioperative harvesting. Various local anesthetics are commonly applied during fat grafting procedures. These substances are known to impair cellular viability, which would affect graft survival and final outcomes, but the exact extent of their impact on ASC biology is unknown. The authors analyzed the short- and long-term effects of lidocaine, mepivacaine, ropivacaine, and bupivacaine at increasing concentrations (0.1 to 10 mM) on primary human ASC proliferation and metabolic activity. Trilinear differentiation was assessed by oil red O stain (adipogenesis), safranin O (chondrogenesis), and cresolphthalein (osteogenesis) labeling. In supernatants, cytokine [interleukin (IL)-6/IL-8, vascular endothelial growth factor, hepatocyte growth factor] secretion was analyzed by enzyme-linked immunosorbent assay. Bupivacaine at greater than 100 µM demonstrated the strongest anti proliferative effects, whereas lidocaine and ropivacaine did not affect cell numbers. Mepivacaine evoked reciprocal results regarding cell count at greater than 1 mM. Each compound impaired trilinear differentiation. Secretion of hepatocyte growth factor and IL-8 was reduced significantly by local anesthetic exposure; levels were restored after substances were washed out. In vitro data show that lidocaine, mepivacaine, and ropivacaine could be applied at concentrations of 1 to 10 mM without affecting ASC survival. In contrast, bupivacaine at concentrations greater than 100 µM should be administered with great caution. The differentiation of ASCs and the ASC's secretome might already be decreased by each local anesthetic at 1 mM. The authors' experimental data can be of great significance to the clinical practice, as local anesthetics are routinely administered during liposuction as a tumescent anesthesia adjunct. Therapeutic, V.

Hepatocyte growth factor-mediated apoptosis mechanisms of cytotoxic CD8+ T cells in normal and cirrhotic livers

Intrahepatic stem/progenitor cells and cytotoxic CD8+ T cells (CD8+ T cells) in the cirrhotic liver undergo apoptosis, which potentially facilitates progression to cancer. Here, we report that hepatocyte growth factor (HGF) signaling plays an important role in promoting normal and damaged liver CD8+ T cell Fas-mediated apoptosis through its only receptor, c-Met. In addition to binding with HGF, c-Met also binds to Fas to form a complex. Using a diethylnitrosamine (DEN)-induced liver fibrosis/cirrhosis mouse model, immunostaining, and terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) staining, we found that HGF secretion was significantly higher at 10 weeks post-DEN, the liver cirrhotic phase (LCP), than at 3 weeks post-DEN, the liver fibrotic phase (LFP). Correspondingly, differences in CD8+ T cell proliferation and apoptosis were noted between the two phases. Interestingly, staining and TUNEL assays revealed lower smooth muscle actin (α-SMA)+ cell apoptosis, a marker for hepatic stellate cells (HSCs), in the LFP group than in the LCP group, which suggested a beneficial correlation among HGF, CD8+ T cells and HSCs in improving the fibrotic load during damaged liver repair. In cultures, when met different concentrations of recombinant HGF (rHGF), phytohemagglutinin (PHA)-stimulated naive mouse splenic CD8+ T cells (pn-msCD8+ T cells) responded differently; as increases in rHGF increased were associated with decreases in the clonal numbers of pn-msCD8+ T cells, and when the rHGF dose was greater than 200 ng/mL, the clonal numbers significantly decreased. In the presence of 400 ng/mL rHGF, the death-inducing signaling complex (DISC) can be directly activated in both nsCD8+ T cells and healthy human peripheral blood CD8+ T cells (hp-CD8+ T cells), as indicated by recruitment of FADD and caspase-8 because DISC forms via the recruitment of FADD and caspase-8, among others. These findings suggest that Fas-mediated apoptosis, may also indicate a regulatory role of HGF signaling in hepatic homeostasis.

Enhanced secretion of hepatocyte growth factor in human umbilical cord mesenchymal stem cells ameliorates pulmonary fibrosis induced by bleomycin in rats.

Umbilical cord mesenchymal stem cells (UCMSCs) are a reportedly promising choice in the treatment of irreversible pulmonary fibrosis and lethal interstitial lung disease with limited drug treatment options. In this study, we investigated the therapeutic efficacy of UCMSCs overexpressing hepatocyte growth factor (HGF), which is considered one of the main anti-fibrotic factors secreted by MSCs. Adenovirus vector carrying the HGF gene was transfected into UCMSCs to produce HGF-modified UCMSCs (HGF-UCMSCs). Transfection promoted the proliferation of UCMSCs and did not change the morphology, and differentiation ability, or biomarkers. Rats were injected with HGF-UCMSCs on days 7 and 11 after intratracheal administration of bleomycin (10mg/kg). We performed an analysis of histopathology and lung function to evaluate the anti-fibrotic effect. The results showed that HGF-UCMSCs decreased the Ashcroft scores in hematoxylin and eosin-stained sections, the percentage positive area in Masson trichrome-stained sections, and the hydroxyproline level in lungs. Forced expiratory volume in the first 300m/forced vital capacity was also improved by HGF-UCMSCs. To explore the possible therapeutic mechanism of HGF-UCMSCs, we detected inflammatory factors in the lungs and performed mRNA sequencing in UCMSCs and HGF-UCMSCs. The data indicated that inhibition of interleukin-17 in the lung may be related to the anti-fibrosis of HGF-UCMSCs, and overexpressed HGF probably played a primary role in the treatment. Collectively, our study findings suggested that the overexpression of HGF may improve the anti-fibrotic effect of UCMSCs through directly or indirectly interacting with interleukin-17-producing cells in fibrotic lungs.

Canine bone marrow peri-adipocyte cells could therapeutically benefit acute spinal cord injury through migration and secretion of hepatocyte growth factor to inflammatory milieu.

Spinal cord injury (SCI) is a common neurological disorder in dogs. A secondary injury that occurs in the acute phase causes expansion of inflammation, resulting in lesion extension and further loss of function. Mesenchymal stem cells (MSCs) have trophic effects and the ability to migrate toward injured tissues; therefore, MSC-based therapy is considered promising for the treatment of canine SCI. We recently reported that bone marrow peri-adipocyte cells (BM-PACs) can be obtained from canine bone marrow and have stem cell potential superior to that of conventional bone marrow MSCs (BMMSCs). However, their therapeutic potential for SCI have been still unknow. Here, we first evaluated the ability of BM-PACs to secrete hepatocyte growth factor (HGF) and their migration ability toward inflammatory milieu in vitro. BM-PACs can secrete HGF in response to pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-α and IL-1β, and exhibit migration ability toward these cytokines. Next, BM-PACs were intravenously administered into nude mice with acute SCI to analyze the homing ability and therapeutic effects of HGF secreted by BM-PACs. BM-PACs homed to the injured spinal cord, where the HGF expression level increased 7 days after administration. Intravenous administration of BM-PACs induced functional recovery and pathological improvement, indicated by less demyelinating area, more preserved axons, and less glial scar formation compared with the mice only received vehicle. These findings suggest that the intravenous administration of BM-PACs can be a novel therapeutic intervention for acute canine SCI.

Combinational Treatment Involving Decellularized Extracellular Matrix Hydrogels With Mesenchymal Stem Cells Increased the Efficacy of Cell Therapy in Pancreatitis.

Cell transplantation using mesenchymal stem cells (MSCs) has emerged as a promising approach to repairing and regenerating injured or impaired organs. However, the survival and retention of MSCs following transplantation remain a challenge. Therefore, we investigated the efficacy of co-transplantation of MSCs and decellularized extracellular matrix (dECM) hydrogels, which have high cytocompatibility and biocompatibility. The dECM solution was prepared by enzymatic digestion of an acellular porcine liver scaffold. It could be gelled and formed into porous fibrillar microstructures at physiological temperatures. MSCs expanded three-dimensionally in the hydrogel without cell death. Compared to the 2-dimensional cell culture, MSCs cultured in the hydrogel showed increased secretion of hepatocyte growth factor (HGF) and tumor necrosis factor-inducible gene 6 protein (TSG-6), both of which are major anti-inflammatory and anti-fibrotic paracrine factors of MSCs, under TNFα stimulation. In vivo experiments showed that the co-transplantation of MSCs with dECM hydrogel improved the survival rate of engrafted cells compared to those administered without the hydrogel. MSCs also demonstrated therapeutic effects in improving inflammation and fibrosis of pancreatic tissue in a dibutyltin dichloride (DBTC)-induced rat pancreatitis model. Combinational use of dECM hydrogel with MSCs is a new strategy to overcome the challenges of cell therapy using MSCs and can be used for treating chronic inflammatory diseases in clinical settings.

Cancer-Associated Fibroblasts Promote Radioresistance of Breast Cancer Cells via the HGF/c-Met Signaling Pathway

Cancer-associated fibroblasts (CAFs) are an integral part of the tumor microenvironment (TME), which is involved in therapy resistance. This study aimed to investigate the role of CAFs in radiosensitivity of breast cancer cells. The CAFs were isolated from the breast cancer tissues, and the conditioned medium was collected to culture breast cancer cells. Radiation-induced DNA damage was evaluated by immunofluorescence and western blotting. Cytokine array and enzyme-linked immunosorbent assay were performed to analyze the secretion of cytokines and growth factors. An in vitro clonogenic survival assay and in vivo xenograft mouse model were performed to determine the radiosensitivity of breast cancer cells. Finally, the expression of hepatocyte growth factor (HGF) and c-Met in the breast cancer tissues were detected by immunohistochemistry. The CAFs were found to secrete HGF to activate the c-Met signaling pathway, which induced epithelial-to-mesenchymal transition, growth, and radioresistance of breast cancer cells. Furthermore, radiation was observed to enhance HGF secretion by CAFs and increase c-Met expression in breast cancer cells, which led to HGF/c-Met signaling pathway activation. Moreover, radiation-induced tumor necrosis factor α (TNFα) secretion by breast cancer cells promoted CAF proliferation and HGF secretion. Additionally, HGF and c-Met high expression were associated with worse recurrence-free survival in patients with breast cancer who had received radiation therapy. The findings revealed that HGF and TNFα are critical for the crosstalk between breast cancer cells and CAFs in the TME and that the HGF/c-Met signaling pathway is a promising therapeutic target for radiosensitizing breast cancer.