Within the tumor microenvironment (TME), cancer cells use mechanotransduction pathways to convert biophysical forces to biochemical signals. However, the underlying mechanisms and functional... Show moreWithin the tumor microenvironment (TME), cancer cells use mechanotransduction pathways to convert biophysical forces to biochemical signals. However, the underlying mechanisms and functional significance of these pathways remain largely unclear. The upregulation of mechanosensitive pathways from biophysical forces such as interstitial flow (IF), leads to the activation of various cytokines, including transforming growth factor-β (TGF-β). TGF-β promotes in part via a Smad-dependent signaling pathway the epithelial–mesenchymal transition (EMT) in cancer cells. The latter process is linked to increased cancer cell motility and invasion. Current research models have limited ability to investigate the combined effects of biophysical forces (such as IF) and cytokines (TGF-β) in a 3D microenvironment. We used a 3D-matrix based microfluidic platform to demonstrate the potentiating effect of IF on exogenous TGF-β induced upregulation of the Smad-signaling activity and the expression of mesenchymal marker vimentin in A549 lung cancer spheroids. To monitor this, we used stably integrated fluorescent based reporters into the A549 cancer cell genome. Our results demonstrate that IF enhances exogenous TGF-β induced Smad-signaling activity in lung cancer spheroids embedded in a matrix microenvironment. In addition, we observed an increased cell motility for A549 spheroids when exposed to IF and TGF-β. Our 3D-microfluidic model integrated with real-time imaging provides a powerful tool for investigating cancer cell signaling and motility associated with invasion characteristics in a physiologically relevant TME. Show less
Crosstalk between cancer and stellate cells is pivotal in pancreatic cancer, resulting in differentiation of stellate cells into myofibroblasts that drives tumour progression. To assess... Show moreCrosstalk between cancer and stellate cells is pivotal in pancreatic cancer, resulting in differentiation of stellate cells into myofibroblasts that drives tumour progression. To assess cooperative mechanisms in a 3D context, we generated chimeric spheroids using human and mouse cancer and stellate cells. Species-specific deconvolution of bulk-RNA sequencing data revealed cell type-specific transcriptomes underpinning invasion. This dataset highlighted stellate-specific expression of transcripts encoding the collagen-processing enzymes ADAMTS2 and ADAMTS14. Strikingly, loss of ADAMTS2 reduced, while loss of ADAMTS14 promoted, myofibroblast differentiation and invasion independently of their primary role in collagen-processing. Functional and proteomic analysis demonstrated that these two enzymes regulate myofibroblast differentiation through opposing roles in the regulation of transforming growth factor β availability, acting on the protease-specific substrates, Serpin E2 and fibulin 2, for ADAMTS2 and ADAMTS14, respectively. Showcasing a broader complexity for these enzymes, we uncovered a novel regulatory axis governing malignant behaviour of the pancreatic cancer stroma. Show less
Gao, Z.C.; Houthuijzen, J.M.; Dijke, P. ten; Brazil, D.P. 2023
GREMLIN1 (GREM1) is member of a family of structurally and functionally related secreted cysteine knot proteins, which act to sequester and inhibit the action of multifunctional bone morphogenetic... Show moreGREMLIN1 (GREM1) is member of a family of structurally and functionally related secreted cysteine knot proteins, which act to sequester and inhibit the action of multifunctional bone morphogenetic proteins (BMPs). GREM1 binds directly to BMP dimers, thereby preventing BMP-mediated activation of BMP type I and type II receptors. Multiple reports identify the overexpression of GREM1 as a contributing factor in a broad range of cancers. Additionally, the GREM1 gene is amplified in a rare autosomal dominant inherited form of colorectal cancer. The inhibitory effects of GREM1 on BMP signaling have been linked to these tumor-promoting effects, including facilitating cancer cell stemness and the activation of cancer-associated fibroblasts. Moreover, GREM1 has been described to bind and signal to vascular endothelial growth factor receptor (VEGFR) and stimulate angiogenesis, as well as epidermal and fibroblast growth factor receptor (EGFR and FGFR) to elicit tumor-promoting effects in breast and prostate cancer, respectively. In contrast, a 2022 report revealed that GREM1 can promote an epithelial state in pancreatic cancers, thereby inhibiting pancreatic tumor growth and metastasis. In this commentary, we will review these disparate findings and attempt to provide clarity around the role of GREM1 signaling in cancer. Show less
Long-lived parasites evade host immunity through highly evolved molecular strategies. The murine intestinal helminth, Heligmosomoides polygyrus, down-modulates the host immune system through... Show moreLong-lived parasites evade host immunity through highly evolved molecular strategies. The murine intestinal helminth, Heligmosomoides polygyrus, down-modulates the host immune system through release of an immunosuppressive TGF-β mimic, TGM1, which is a divergent member of the CCP (Sushi) protein family. TGM1 comprises 5 domains, of which domains 1-3 (D1/2/3) bind mammalian TGF-β receptors, acting on T cells to induce Foxp3+ regulatory T cells; however, the roles of domains 4 and 5 (D4/5) remain unknown. We noted that truncated TGM1, lacking D4/5, showed reduced potency. Combination of D1/2/3 and D4/5 as separate proteins did not alter potency, suggesting that a physical linkage is required and that these domains do not deliver an independent signal. Coprecipitation from cells treated with biotinylated D4/5, followed by mass spectrometry, identified the cell surface protein CD44 as a coreceptor for TGM1. Both full-length and D4/5 bound strongly to a range of primary cells and cell lines, to a greater degree than D1/2/3 alone, although some cell lines did not respond to TGM1. Ectopic expression of CD44 in nonresponding cells conferred responsiveness, while genetic depletion of CD44 abolished enhancement by D4/5 and ablated the ability of full-length TGM1 to bind to cell surfaces. Moreover, CD44-deficient T cells showed attenuated induction of Foxp3 by full-length TGM1, to levels similar to those induced by D1/2/3. Hence, a parasite protein known to bind two host cytokine receptor subunits has evolved a third receptor specificity, which serves to raise the avidity and cell type–specific potency of TGF-β signaling in mammalian cells. Show less
The cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway is a promising approach for anti-cancer immunotherapy by bridging innate and adaptive immunity. Recent evidence... Show moreThe cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway is a promising approach for anti-cancer immunotherapy by bridging innate and adaptive immunity. Recent evidence suggests that chemotherapy-induced DNA damage can directly induce dendritic cell (DC) maturation and recruitment, which synergizes with STING activation to enhance anti-tumor effects. As an immunogenic cell death (ICD) inducer, oxaliplatin generates massive double-stranded DNA (dsDNA) crosslinks, release of tumor-associated antigens and promoting the "eat me" signal. STING activation improves antigen immunogenicity, which can promote T cell activation and infiltration. In this study, we developed liposomes encapsulating oxaliplatin and combine this formulation with a STING agonist (ADU-S100) for treating colorectal cancer. The liposomes efficiently inhibited the proliferation of tumor cells while induced ICD in CT26 colorectal cancer cells, which enhanced dendritic cell maturation and phagocytosis in vitro. The liposome-based immunochemotherapy exhibited the strongest efficacy, resulting in complete remission upon tumor inoculation. Mechanistic studies showed this potent anti-cancer effect was related to the significant recruitment of infiltrating CD8 and CD4 T cells, reduction of suppressive Treg cells, and a shift in the phenotype of tumor-associated suppressive macrophages that promote cancer to immune stimulating macrophages. Thus, our study demonstrated the potential of combining oxaliplatin-loaded liposomes with a STING agonist to reduce tumor growth by regulating the immunosuppressive state in the tumor. Show less
Liu, S.J.; Dinther, M. van; Hagenaars, S.C.; Gu, Y.Z.; Kuipers, T.B.; Mei, H.L.; ... ; Dijke, P. ten 2023
Triple-negative breast cancer (TNBC) is the most challenging breast cancer subtype to treat due to its aggressive characteristics and low response to the existing clinical therapies. Distant... Show moreTriple-negative breast cancer (TNBC) is the most challenging breast cancer subtype to treat due to its aggressive characteristics and low response to the existing clinical therapies. Distant metastasis is the main cause of death of TNBC patients. Better understanding of the mechanisms underlying TNBC metastasis may lead to new strategies of early diagnosis and more efficient treatment. In our study, we uncovered that the autophagy receptor optineurin (OPTN) plays an unexpected role in TNBC metastasis. Data mining of publicly available data bases revealed that the mRNA level of OPTN in TNBC patients positively correlates with relapse free and distance metastasis free survival. Importantly, in vitro and in vivo models demonstrated that OPTN suppresses TNBC metastasis. Mechanistically, OPTN inhibited the pro-oncogenic transforming growth factor-beta (TGF beta) signaling in TNBC cells by interacting with TGF beta type I receptor (T beta RI) and promoting its ubiquitination for degradation. Consistent with our experimental findings, the clinical TNBC samples displayed a negative correlation between OPTN mRNA expression and TGF beta gene response signature and expression of proto-typic TGF beta target genes. Altogether, our study demonstrates that OPTN is a negative regulator for TGF beta receptor/SMAD signaling and suppresses metastasis in TNBC. Show less
Zhang, J.; Dinther, M. van; Thorikay, M.; Gourabi, B.M.; Kruithof, B.P.T.; Dijke, P. ten 2023
Ubiquitin-specific protease (USP)19 is a deubiquitinating enzyme that regulates the stability and function of multiple proteins, thereby controlling various biological responses. The alternative... Show moreUbiquitin-specific protease (USP)19 is a deubiquitinating enzyme that regulates the stability and function of multiple proteins, thereby controlling various biological responses. The alternative splicing of USP19 results in the expression of two major encoded variants that are localized to the endoplasmic reticulum (ER) (USP19-ER) and cytoplasm (USP19-CY). The importance of alternative splicing for the function of USP19 remains unclear. Here, we demonstrated that USP19-CY promotes TGF-beta signaling by directly interacting with TGF-beta type I receptor (T beta RI) and protecting it from degradation at the plasma membrane. In contrast, USP19-ER binds to and sequesters T beta RI in the ER. By decreasing cell surface T beta RI levels, USP19-ER inhibits TGF-beta/SMAD signaling in a deubiquitination-independent manner. Moreover, USP19-ER inhibits TGF-beta-induced epithelial-mesenchymal transition (EMT), whereas USP19-CY enhances EMT, as well as the migration and extravasation of breast cancer cells. Furthermore, USP19-CY expression is correlated with poor prognosis and is higher in breast cancer tissues than in adjacent normal tissues. Notably, the splicing modulator herboxidiene inhibits USP19-CY, increases USP19-ER expression and suppresses breast cancer cell migration. Targeting USP19 splicing or its deubiquitinating activity may have potential therapeutic effects on breast cancer. Show less
Zhang, J.; Zon, G. van der; Ma, J.; Mei, H.L.; Cabukusta, B.; Agaser, C.C.; ... ; Dijke, P. ten 2022
Epithelial-mesenchymal transition (EMT) is pivotal in the initiation and development of cancer cell metastasis. We observed that the abundance of glycosphingolipids (GSLs), especially ganglioside... Show moreEpithelial-mesenchymal transition (EMT) is pivotal in the initiation and development of cancer cell metastasis. We observed that the abundance of glycosphingolipids (GSLs), especially ganglioside subtypes, decreased significantly during TGF-beta-induced EMT in NMuMG mouse mammary epithelial cells and A549 human lung adenocarcinoma cells. Transcriptional profiling showed that TGF-beta/SMAD response genes and EMT signatures were strongly enriched in NMuMG cells, along with depletion of UDP-glucose ceramide glucosyltransferase (UGCG), the enzyme that catalyzes the initial step in GSL biosynthesis. Consistent with this finding, genetic or pharmacological inhibition of UGCG promoted TGF-beta signaling and TGF-beta-induced EMT. UGCG inhibition promoted A549 cell migration, extravasation in the zebrafish xenograft model, and metastasis in mice. Mechanistically, GSLs inhibited TGF-beta signaling by promoting lipid raft localization of the TGF-beta type I receptor (T beta RI) and by increasing T beta RI ubiquitination and degradation. Importantly, we identified ST3GAL5-synthesized a-series gangliosides as the main GSL subtype involved in inhibition of TGF-beta signaling and TGF-beta-induced EMT in A549 cells. Notably, ST3GAL5 is weakly expressed in lung cancer tissues compared to adjacent nonmalignant tissues, and its expression correlates with good prognosis. Show less
Yan, X.H.; Zhang, L.; Miyazawa, K.; Dijke, P. ten 2022
Photodynamic therapy (PDT) has shown impressive therapeutic effects on various types of cancers by reactive oxygen species (ROS) generation and induction of immune responses. However, under certain... Show morePhotodynamic therapy (PDT) has shown impressive therapeutic effects on various types of cancers by reactive oxygen species (ROS) generation and induction of immune responses. However, under certain conditions, the immune responses induced by PDT are not always sufficient to eradicate the remaining tumor cells. On the other hand, the photosensitizer indocyanine green (ICG) can mediate PDT under near-infrared (NIR) illumination, thereby enhancing the penetration depth of the excitation light into the tumor. We found that ICG is rapidly taken up in vitro by colorectal MC38 and CT26 tumor cells and it promotes PDT-mediated cell-killing effects. Our results furthermore revealed that ICG induces immunogenic cell death (ICD), as dendritic cells (DCs) were found to engulf ICG-PDT-treated tumor cells and undergo phenotypic maturation. ICG accumulated in tumors 2 h after administration, as measured by fluorescence and photoacoustic imaging. Considering the advantages of ICG as a photosensitizer, we sought to design a therapy that combines PDT and immune checkpoint blockade to maximize tumor control. To this end, a 25% thermosensitive polymer 407 hydrogel was included as a co-delivery platform for this treatment scheme. NIR-PDT under 808 nm irradiation in combination with cytotoxic T-lymphocyte-associated protein 4 (CTLA4)/programmed death-ligand 1 (PD-L1) checkpoint blockade prolonged survival rate of colorectal tumor-bearing mice by inducing a series of immune responses, like the phagocytosis of tumor debris by macrophages and DCs, and induction of acute inflammation, leukocyte infiltration, maturation and activation of DCs. Altogether, our work presents a NIR-triggered PDT strategy in combination with immune checkpoint blockade. Compared to a single treatment, the combination treatment increased efficiency to inhibit solid tumor growth and improved the survival rate of tumor-bearing mice. Show less
Xie, F.; Zhou, X.X.; Su, P.; Li, H.Y.; Tu, Y.F.; J.J. du; ... ; Zhou, F.F. 2022
Understanding the factors that hamper immune therapy in breast cancer may increase the range of patients who benefit. Here authors show that breast cancer cells produce and subsequently transfer... Show moreUnderstanding the factors that hamper immune therapy in breast cancer may increase the range of patients who benefit. Here authors show that breast cancer cells produce and subsequently transfer active TGF-beta type II receptors to CD8 + T cells to render them exhausted, thus paralyzing the anti-tumor immune response.Cancer immunotherapies have shown clinical success in various types of tumors but the patient response rate is low, particularly in breast cancer. Here we report that malignant breast cancer cells can transfer active TGF-beta type II receptor (T beta RII) via tumor-derived extracellular vesicles (TEV) and thereby stimulate TGF-beta signaling in recipient cells. Up-take of extracellular vesicle-T beta RII (EV-T beta RII) in low-grade tumor cells initiates epithelial-to-mesenchymal transition (EMT), thus reinforcing cancer stemness and increasing metastasis in intracardial xenograft and orthotopic transplantation models. EV-T beta RII delivered as cargo to CD8(+) T cells induces the activation of SMAD3 which we demonstrated to associate and cooperate with TCF1 transcription factor to impose CD8(+) T cell exhaustion, resulting in failure of immunotherapy. The levels of T beta RII+ circulating extracellular vesicles (crEV) appears to correlate with tumor burden, metastasis and patient survival, thereby serve as a non-invasive screening tool to detect malignant breast tumor stages. Thus, our findings not only identify a possible mechanism by which breast cancer cells can promote T cell exhaustion and dampen host anti-tumor immunity, but may also identify a target for immune therapy against the most devastating breast tumors. Show less
Mehta, P.; Rahman, Z.; Dijke, P. ten; Boukany, P.E. 2022
An early step of metastasis requires a complex and coordinated migration of invasive tumor cells into the surrounding tumor microenvironment (TME), which contains extracellular matrix (ECM). It is... Show moreAn early step of metastasis requires a complex and coordinated migration of invasive tumor cells into the surrounding tumor microenvironment (TME), which contains extracellular matrix (ECM). It is being appreciated that 3D matrix -based microfluidic models have an advantage over conventional in vitro and animal models to study tumor progression events. Recent microfluidic models have enabled recapitulation of key mechanobiological features present within the TME to investigate collective cancer cell migration and invasion. Microfluidics also allows for functional interrogation and therapeutic manipulation of specific steps to study the dynamic aspects of tumor progression. In this review, we focus on recent developments in cancer cell migration and how microfluidic strategies have evolved to address the physiological complexities of the TME to visualize migration modes adapted by various tumor cells. Show less
Background and Aims: In hereditary hemorrhagic telangiectasia (HHT), severe liver vascular malformations are associated with mutations in the Activin A Receptor-Like Type 1 (ACVRL1) gene encoding... Show moreBackground and Aims: In hereditary hemorrhagic telangiectasia (HHT), severe liver vascular malformations are associated with mutations in the Activin A Receptor-Like Type 1 (ACVRL1) gene encoding ALK1, the receptor for bone morphogenetic protein (BMP) 9/BMP10, which regulates blood vessel development. Here, we established an HHT mouse model with exclusive liver involvement and adequate life expectancy to investigate ALK1 signaling in liver vessel formation and metabolic function. Approach and Results: Liver sinusoidal endothelial cell (LSEC)-selective Cre deleter line, Stab2-iCreF3, was crossed with Acvrl1-floxed mice to generate LSEC-specific Acvrl1-deficient mice (Alk1(HEC-KO)). Alk1(HEC-KO) mice revealed hepatic vascular malformations and increased posthepatic flow, causing right ventricular volume overload. Transcriptomic analyses demonstrated induction of proangiogenic/tip cell gene sets and arterialization of hepatic vessels at the expense of LSEC and central venous identities. Loss of LSEC angiokines Wnt2, Wnt9b, and R-spondin-3 (Rspo3) led to disruption of metabolic liver zonation in Alk1(HEC-KO) mice and in liver specimens of patients with HHT. Furthermore, prion-like protein doppel (Prnd) and placental growth factor (Pgf) were upregulated in Alk1(HEC-KO) hepatic endothelial cells, representing candidates driving the organ-specific pathogenesis of HHT. In LSEC in vitro, stimulation or inhibition of ALK1 signaling counter-regulated Inhibitors of DNA binding (ID)1-3, known Alk1 transcriptional targets. Stimulation of ALK1 signaling and inhibition of ID1-3 function confirmed regulation of Wnt2 and Rspo3 by the BMP9/ALK1/ID axis. Conclusions: Hepatic endothelial ALK1 signaling protects from development of vascular malformations preserving organ-specific endothelial differentiation and angiocrine signaling. The long-term surviving Alk1(HEC-KO) HHT model offers opportunities to develop targeted therapies for this severe disease. Show less
Orlova, V.V.; Nahon, D.M.; Cochrane, A.; Cao, X.; Freund, C.; Hil, F. van den; ... ; Mummery, C.L. 2022
Hereditary hemorrhagic telangiectasia (HHT) is a genetic disease characterized by weak blood vessels. HHT1 is caused by mutations in the ENDOGLIN (ENG) gene. Here, we generated induced pluripotent... Show moreHereditary hemorrhagic telangiectasia (HHT) is a genetic disease characterized by weak blood vessels. HHT1 is caused by mutations in the ENDOGLIN (ENG) gene. Here, we generated induced pluripotent stem cells (hiPSCs) from a patient with rare mosaic HHT1 with tissues containing both mutant (ENG(c.)(1678C>)(T)) and normal cells, enabling derivation of isogenic diseased and healthy hiPSCs, respectively. We showed reduced ENG expression in HHT1 endothelial cells (HHT1-hiPSC-ECs), reflecting haploinsufficiency. HHT1(c.)(1678C)(>T)-hiPSC-ECs and the healthy isogenic control behaved similarly in two-dimensional (2D) culture, forming functionally indistinguishable vascular networks. However, when grown in 3D organ-on-chip devices under microfluidic flow, lumenized vessels formed in which defective vascular organization was evident: interaction between inner ECs and surrounding pericytes was decreased, and there was evidence for vascular leakage. Organs on chip thus revealed features of HHT in hiPSC-derived blood vessels that were not evident in conventional 2D assays. Show less
Xie, F.; Zhou, X.X.; Li, H.Y.; Su, P.; Liu, S.J.; Li, R.; ... ; Zhang, L. 2022
TGF-beta signaling is a key player in tumor progression and immune evasion, and is associated with poor response to cancer immunotherapies. Here, we identified ubiquitin-specific peptidase 8 (USP8)... Show moreTGF-beta signaling is a key player in tumor progression and immune evasion, and is associated with poor response to cancer immunotherapies. Here, we identified ubiquitin-specific peptidase 8 (USP8) as a metastasis enhancer and a highly active deubiquitinase in aggressive breast tumors. USP8 acts both as a cancer stemness-promoting factor and an activator of the TGF-beta/SMAD signaling pathway. USP8 directly deubiquitinates and stabilizes the type II TGF-beta receptor T beta RII, leading to its increased expression in the plasma membrane and in tumor-derived extracellular vesicles (TEVs). Increased USP8 activity was observed in patients resistant to neoadjuvant chemotherapies. USP8 promotes TGF-beta/SMAD-induced epithelial-mesenchymal transition (EMT), invasion, and metastasis in tumor cells. USP8 expression also enables T beta RII+ circulating extracellular vesicles (crEVs) to induce T cell exhaustion and chemoimmunotherapy resistance. Pharmacological inhibition of USP8 antagonizes TGF-beta/SMAD signaling, and reduces T beta RII stability and the number of T beta RII+ crEVs to prevent CD8+ T cell exhaustion and to reactivate anti-tumor immunity. Our findings not only reveal a novel mechanism whereby USP8 regulates the cancer microenvironment but also demonstrate the therapeutic advantages of engineering USP8 inhibitors to simultaneously suppress metastasis and improve the efficacy of cancer immunotherapy. Show less
Marvin, D.L.; Spaans, V.M.; Kroon, C.D. de; Slieker, R.C.; Khelil, M.; Dijke, P. ten; ... ; Jordanova, E.S. 2022
Cervical cancer is the fourth most common cancer in women worldwide. Squamous cell carcinoma (SCC) and adenocarcinoma (AC) are the most common histological types, with AC patients having worse... Show moreCervical cancer is the fourth most common cancer in women worldwide. Squamous cell carcinoma (SCC) and adenocarcinoma (AC) are the most common histological types, with AC patients having worse prognosis. Over the last two decades, incidence rates of AC have increased, highlighting the importance of further understanding AC tumorigenesis, and the need to investigate new treatment options. The cytokine TGF-beta functions as a tumour suppressor in healthy tissue. However, in tumour cells this suppressive function can be overcome. Therefore there is an increasing interest in using TGF-beta inhibitors in the treatment of cancer. Here, we hypothesize that TGF-beta plays a different role in SCC and AC. Analysis of RNA-seq data from the TCGA, using a TGF-beta response signature, resulted in separate clustering of the two subtypes. We further investigated the expression of TGF-beta-signalling related proteins (T beta R1/2, SMAD4, pSMAD2, PAI-1, alpha v beta 6 and MMP2/9) in a cohort of 62 AC patients. Low T beta R2 and SMAD4 expression was associated with worse survival in AC patients and interestingly, high PAI-1 and alpha v beta 6 expression was also correlated with worse survival. Similar correlations of T beta R2, PAI-1 and alpha v beta 6 with clinical parameters were found in previously reported SCC analyses. However, when comparing expression levels between SCC and AC patient samples, pSMAD2, SMAD4, PAI-1 and alpha v beta 6 showed lower expression in AC compared to SCC. Because of the low expression of core T beta R1/2, (p-)SMAD2 and SMAD4 proteins and the correlation with worse prognosis, TGF-beta pathway most likely leads to tumour inhibitory effects in AC and therefore the use of TGF-beta inhibitors would not be recommended. However, given the correlation of PAI-1 and alpha v beta 6 with poor prognosis, the use of TGF- beta inhibitors might be of interest in SCC and in the subsets of AC patients with high expression of these TGF-beta associated proteins. Show less
Rotteveel, L.; Kurakula, K.; Kooijman, E.J.M.; Schuit, R.C.; Verlaan, M.; Schreurs, M.; ... ; Windhorst, A.D. 2022
The transforming growth factor beta (TGF beta) pathway plays a complex role in cancer biology, being involved in both tumour suppression as well as promotion. Overactive TGF beta signalling has... Show moreThe transforming growth factor beta (TGF beta) pathway plays a complex role in cancer biology, being involved in both tumour suppression as well as promotion. Overactive TGF beta signalling has been linked to multiple diseases, including cancer, pulmonary arterial hypertension, and fibrosis. One of the key meditators within this pathway is the TGF beta type I receptor, also termed activin receptor-like kinase 5 (ALK5). ALK5 expression level is a key determinant of TGF beta signalling intensity and duration, and perturbation has been linked to diseases. A validated ALK5 positron emission tomography (PET) tracer creates an opportunity, therefore, to study its role in human diseases. To develop ALK5 PET tracers, two small molecule ALK5 kinase inhibitors were selected as lead compounds, which were labelled with carbon-11 and fluorine-18, respectively. [C-11]LR111 was synthesized with a yield of 17 +/- 6%, a molar activity of 126 +/- 79 GBq. mol(-1) and a purity of > 95% (n = 44). [18F]EW-7197 was synthesized with a yield of 10 +/- 5%, a molar activity of 183 & PLUSMN; 126 GBq. mol(-1) and a purity of > 95% (n = 11). Metabolic stability was evaluated in vivo in mice, showing 39 +/- 2% of intact [11C]LR111 and 21 +/- 2% of intact [F-18]EW-7197 in blood plasma at 45 min p.i. In vitro binding experiments were conducted in breast cancer MDAMB-231 and lung cancer A431 cell lines. In addition, both tracers were used for PET imaging in MDA-MB-231 xenograft models. Selective uptake of [F-18]EW-7197 and [C-11]LR111 was observed in MDA-MB-231 cells, in the MDA-MB-231 tumour xenografts in vivo and in the autoradiograms. As [C-11]LR111 and [F-18]EW-7197 showed selectivity of binding to ALK5 in vivo and in vitro. Both tracers are thereby valuable tools for the detection of ALK5 activity. Show less
Simple Summary Photodynamic therapy (PDT) by means of a photosensitizer is a clinically used therapeutic treatment in a variety of cancers. To further improve the anti-cancer efficiency of PDT,... Show moreSimple Summary Photodynamic therapy (PDT) by means of a photosensitizer is a clinically used therapeutic treatment in a variety of cancers. To further improve the anti-cancer efficiency of PDT, combination therapy with immune agents is a promising option. In this study, we used a viral vaccine as the immune therapeutic partner for PDT. We studied the biological properties of single and combined modalities. Our research suggests that combination therapy enhances innate and humoral immunity, improved survival, and generated a long-term memory capacity in the MC-38 murine colorectal tumor model to prevent a recurrence. Photodynamic therapy (PDT), which combines light and oxygen with a photosensitizer to induce reactive oxygen species (ROS)-mediated killing of primary tumor cells, benefits from non-invasive properties and its negligible toxicity to surrounding healthy tissues. In this study, we have shown that the second-generation photosensitizer FOSCAN can be internalized by tumor cells and effectively induce tumor cell death when exposed to laser irradiation in vitro. In addition, these dying tumor cells can be phagocytosed by dendritic cells and lead to their activation and maturation as assessed by in vitro co-culture models. While PDT induces immunogenic tumor cell apoptosis, its application for the treatment of tumors located in deep tissues and advanced malignancies has been limited. In this study, we demonstrate that hepatitis B core virus-like particles (HBc VLPs) can serve as a vaccine to enhance PDT-induced anti-cancer immunity by priming humoral immune responses and inducing CD8(+) T cell responses. The combination of PDT and HBc VLPs increased the survival rate of MC-38 tumor-bearing mice to 55%, compared to 33% in PDT alone and no tumor-free mice in vaccine alone. Moreover, the combination effectively prevented tumor recurrence in vivo through enhanced immune memory T cells after therapy. Therefore, as both are clinically approved techniques, this combination provides a promising strategy for cancer therapy. Show less
Guo, J.X.; Liu, B.; Thorikay, M.; Yu, M.M.; Li, X.Y.; Tong, Z.; ... ; Li, W. 2022
Heterozygous mutations in BMPR2 (bone morphogenetic protein (BMP) receptor type II) cause pulmonary arterial hypertension. BMPRII is a receptor for over 15 BMP ligands, but why BMPR2 mutations... Show moreHeterozygous mutations in BMPR2 (bone morphogenetic protein (BMP) receptor type II) cause pulmonary arterial hypertension. BMPRII is a receptor for over 15 BMP ligands, but why BMPR2 mutations cause lung-specific pathology is unknown. To elucidate the molecular basis of BMP:BMPRII interactions, we report crystal structures of binary and ternary BMPRII receptor complexes with BMP10, which contain an ensemble of seven different BMP10:BMPRII 1:1 complexes. BMPRII binds BMP10 at the knuckle epitope, with the A-loop and beta 4 strand making BMPRII-specific interactions. The BMPRII binding surface on BMP10 is dynamic, and the affinity is weaker in the ternary complex than in the binary complex. Hydrophobic core and A-loop interactions are important in BMPRII-mediated signalling. Our data reveal how BMPRII is a low affinity receptor, implying that forming a signalling complex requires high concentrations of BMPRII, hence mutations will impact on tissues with highest BMPR2 expression such as the lung vasculature.Mutations in BMPR2 is the major genetic cause for pulmonary arterial hypertension (PAH). Here by solving crystal structures of BMPRII in binary and ternary receptor complexes with BMP10, the authors report the molecular recognition between BMPRII and BMP10, and its implication in PAH. Show less
Marvin, D.L.; You, L.; Bornes, L.; Dinther, M. van; Peters, N.; Dang, H.; ... ; Ritsma, L. 2022
Transforming growth factor-beta (TGF-beta) signaling is tightly controlled in duration and intensity during embryonic development and in the adult to maintain tissue homeostasis. To visualize the... Show moreTransforming growth factor-beta (TGF-beta) signaling is tightly controlled in duration and intensity during embryonic development and in the adult to maintain tissue homeostasis. To visualize the TGF-beta/SMAD3 signaling kinetics, we developed a dynamic TGF-beta/SMAD3 transcriptional fluorescent reporter using multimerized SMAD3/4 binding elements driving the expression of a quickly folded and highly unstable GFP protein. We demonstrate the specificity and sensitivity of this reporter and its wide application to monitor dynamic TGF-beta/SMAD3 transcriptional responses in both 2D and 3D systems in vitro, as well as in vivo, using live-cell and intravital imaging. Using this reporter in B16F10 cells, we observed single cell heterogeneity in response to TGF-beta challenge, which can be categorized into early, late, and non-responders. Because of its broad application potential, this reporter allows for new discoveries into how TGF-beta/SMAD3-dependent transcriptional dynamics are affected during multistep and reversible biological processes. Show less
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