Protein Activation Research Articles

Modeling the profibrotic microenvironment in vitro: Model validation

Establishing the molecular and cellular mechanisms of fibrosis requires the development of validated and reproducible models. The complexity of in vivo models challenges the monitoring of an individual cell fate, in some cases making it impossible. However, the set of factors affecting cells in vitro culture systems differ significantly from in vivo conditions, insufficiently reproducing living systems. Thus, to model profibrotic conditions in vitro, usually the key profibrotic factor, transforming growth factor beta (TGFβ-1) is used as a single factor. TGFβ-1 stimulates the differentiation of fibroblasts into myofibroblasts, the main effector cells promoting the development and progression of fibrosis. However, except for soluble factors, the rigidity and composition of the extracellular matrix (ECM) play a critical role in the differentiation process. To develop the model of more complex profibrotic microenvironment in vitro, we used a combination of factors: decellularized ECM synthesized by human dermal fibroblasts in the presence of ascorbic acid if cultured as cell sheets and recombinant TGFβ-1 as a supplement. When culturing human mesenchymal stromal cells derived from adipose tissue (MSCs) under described conditions, we observed differentiation of MSCs into myofibroblasts due to increased number of cells with stress fibrils with alpha-smooth muscle actin (αSMA), and increased expression of myofibroblast marker genes such as collagen I, EDA-fibronectin and αSMA. Importantly, secretome of MSCs changed in these profibrotic microenvironment: the secretion of the profibrotic proteins SPARC and fibulin-2 increased, while the secretion of the antifibrotic hepatocyte growth factor (HGF) decreased. Analysis of transciptomic pattern of regulatory microRNAs in MSCs revealed 49 miRNAs with increased expression and 3 miRNAs with decreased expression under profibrotic stimuli. Bioinformatics analysis confirmed that at least 184 gene targets of the differently expressed miRNAs genes were associated with fibrosis. To further validate the developed model of profibrotic microenvironment, we cultured human dermal fibroblasts in these conditions and observed increased expression of fibroblast activation protein (FAPa) after 12 h of cultivation as well as increased level of αSMA and higher number of αSMA + stress fibrils after 72 h. The data obtained allow us to conclude that the conditions formed by the combination of profibrotic ECM and TGFβ-1 provide a complex profibrotic microenvironment in vitro. Thus, this model can be applicable in studying the mechanism of fibrosis development, as well as for the development of antifibrotic therapy.

Magnetic Resonance Imaging of Fibroblast Activation Protein Using a Targeted Gadolinium-Based Contrast Agent.

The aim of this study was to synthesize a quinoline-based MRI contrast agent, Gd-DOTA-FAPI04, and assess its capacity for targeting fibroblast activation protein (FAP)-positive tumors in vivo. Gd-DOTA-FAPI04 was synthesized by attaching a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) complex of gadolinium(III) to FAP inhibitor FAPI04. The longitudinal relaxation time (T1) of the contrast agent was measured using a Siemens Prisma 3.0T MR system, and the CCK-8 assay was performed to evaluate its potential cytotoxicity. Male nude mice bearing tumors grown from FAP-expressing fibrosarcoma cells were divided into experimental (n = 4) and control (n = 4) groups, and T1-weighted image enhancement was measured at different times (0, 10, 30, 60, 90, and 120 min) postinjection of Gd-DOTA-FAPI04. The control group received an additional preinjection of excess FAPI04. FAP expression in tumor tissue was investigated by using immunohistochemistry with an anti-FAP antibody. The longitudinal relaxivities of gadodiamide and Gd-DOTA-FAPI04 were measured to be 3.734 mM-1 s-1 and 5.323 mM-1 s-1, respectively. The CCK-8 assay demonstrated that Gd-DOTA-FAPI04 has minimal toxicity to cultured human fibrosarcoma cells. In vivo MRI showed that peak accumulation of Gd-DOTA-FAPI04 in FAP-expressing tumors occurred 1 h postinjection and could be blocked by preinjection of excess FAPI04. Immunohistochemical analysis of harvested tumor tissue supported the above findings. Gd-DOTA-FAPI04 is a promising contrast agent for in vivo imaging of FAP.

Cyclase-associated protein (CAP) inhibits inverted formin 2 (INF2) to induce dendritic spine maturation

The morphology of dendritic spines, the postsynaptic compartment of most excitatory synapses, decisively modulates the function of neuronal circuits as also evident from human brain disorders associated with altered spine density or morphology. Actin filaments (F-actin) form the backbone of spines, and a number of actin-binding proteins (ABP) have been implicated in shaping the cytoskeleton in mature spines. Instead, only little is known about the mechanisms that control the reorganization from unbranched F-actin of immature spines to the complex, highly branched cytoskeleton of mature spines. Here, we demonstrate impaired spine maturation in hippocampal neurons upon genetic inactivation of cyclase-associated protein 1 (CAP1) and CAP2, but not of CAP1 or CAP2 alone. We found a similar spine maturation defect upon overactivation of inverted formin 2 (INF2), a nucleator of unbranched F-actin with hitherto unknown synaptic function. While INF2 overactivation failed in altering spine density or morphology in CAP-deficient neurons, INF2 inactivation largely rescued their spine defects. From our data we conclude that CAPs inhibit INF2 to induce spine maturation. Since we previously showed that CAPs promote cofilin1-mediated cytoskeletal remodeling in mature spines, we identified them as a molecular switch that control transition from filopodia-like to mature spines.

A highly sensitive reporter system to monitor endogenous YAP1/TAZ activity and its application in various human cells.

The activation of yes-associated protein 1 (YAP1) and transcriptional co-activator with PDZ-binding motif (TAZ) has been implicated in both regeneration and tumorigenesis, thus representing a double-edged sword in tissue homeostasis. However, how the activity of YAP1/TAZ is regulated or what leads to its dysregulation in these processes remains unknown. To explore the upstream stimuli modulating the cellular activity of YAP1/TAZ, we developed a highly sensitive YAP1/TAZ/TEAD-responsive DNA element (YRE) and incorporated it into a lentivirus-based reporter cell system to allow for sensitive and specific monitoring of the endogenous activity of YAP1/TAZ in terms of luciferase activity invitro and Venus fluorescence invivo. Furthermore, by replacing YRE with TCF- and NF-κB-binding DNA elements, we demonstrated the applicability of this reporter system to other pathways such as Wnt/β-catenin/TCF- and IL-1β/NF-κB-mediated signaling, respectively. The practicality of this system was evaluated by performing cell-based reporter screening of a chemical compound library consisting of 364 known inhibitors, using reporter-introduced cells capable of quantifying YAP1/TAZ- and β-catenin-mediated transcription activities, which led to the identification of multiple inhibitors, including previously known as well as novel modulators of these signaling pathways. We further confirmed that novel YAP1/TAZ modulators, such as potassium ionophores, Janus kinase inhibitors, platelet-derived growth factor receptor inhibitors, and genotoxic stress inducers, alter the protein level or phosphorylation of endogenous YAP1/TAZ and the expression of their target genes. Thus, this reporter system provides a powerful tool to monitor endogenous signaling activities of interest (even in living cells) and search for modulators in various cellular contexts.

Harnessing the Potential of FAP-IL-12mut TMEkine™ for Targeted and Enhanced Anti-tumor Responses.

While cancer immunotherapy has yielded encouraging outcomes in hematological malignancies, it has faced challenges in achieving the same level of effectiveness in numerous solid tumors, primarily because of the presence of immune-suppressive tumor microenvironments (TMEs). The immunosuppressive qualities of the TME have generated considerable interest, making it a focal point for treatments aimed at enhancing immune responses and inhibiting tumor progression. Fibroblast activation protein (FAP), an attractive candidate for targeted immunotherapy, is prominently expressed in the TME of various solid tumors. Interleukin-12 (IL-12), recognized as a key mediator of immune responses, has been explored as a potential candidate for cancer treatment. Nevertheless, initial efforts to administer IL-12 systemically demonstrated limited efficacy and notable side effects, emphasizing the necessity for innovation. To address these concerns, our molecules incorporated specific IL-12 mutations, called IL-12mut, which reduced toxicity. This study explored the therapeutic potential of the FAP-IL-12mut TMEkine™-a novel immunotherapeutic agent selectively engineered to target FAP-expressing cells in preclinical cancer models. Our preclinical results, conducted across diverse murine cancer models, demonstrated that FAP-IL-12mut significantly inhibits tumor growth, enhances immune cell infiltration, and promotes a shift toward a cytotoxic immune activation profile. These findings suggest that FAP-IL-12mut could offer effective cancer treatment strategies.

Involvement of lysophosphatidic acid-LPA1-YAP signaling in healthy and pathological FAPs migration

Skeletal muscle fibrosis is defined as the excessive accumulation of extracellular matrix (ECM) components and is a hallmark of muscular dystrophies. Fibro-adipogenic progenitors (FAPs) are the main source of ECM, and thus have been strongly implicated in fibrogenesis. In skeletal muscle fibrotic models, including muscular dystrophies, FAPs undergo dysregulations in terms of proliferation, differentiation, and apoptosis, however few studies have explored the impact of FAPs migration.Here, we studied fibroblast and FAPs migration and identified lysophosphatidic acid (LPA), a signaling lipid central to skeletal muscle fibrogenesis, as a significant migration inductor. We identified LPA receptor 1 (LPA1) mediated signaling as crucial for this effect through a mechanism dependent on the Hippo pathway, another pathway implicated in fibrosis across diverse tissues. This cross-talk favors the activation of the Yes-associated protein 1 (YAP) and Transcriptional coactivator with PDZ-binding motif (TAZ), leading to increased expression of fibrosis-associated genes. This study reveals the role of YAP in LPA-mediated fibrotic responses as inhibition of YAP transcriptional coactivator activity hinders LPA-induced migration in fibroblasts and FAPs.Moreover, we found that FAPs derived from the mdx4cv mice, a murine model of Duchenne muscular dystrophy, display a heightened migratory phenotype due to enhanced LPA signaling compared to wild-type FAPs. Remarkably, we found that the inhibition of LPA1 or YAP transcriptional coactivator activity in mdx4cv FAPs reverts this phenotype. In summary, the identified LPA-LPA1-YAP pathway emerges as a critical driver of skeletal muscle FAPs migration and provides insights into potential novel targets to mitigate fibrosis in muscular dystrophies.

Erratum for the Research Article "A β-Arrestin-Biased Agonist of the Parathyroid Hormone Receptor (PTH1R) Promotes Bone Formation Independent of G Protein Activation" by D. Gesty-Palmer et al.

Erratum for the Research Article "A β-Arrestin-Biased Agonist of the Parathyroid Hormone Receptor (PTH1R) Promotes Bone Formation Independent of G Protein Activation" by D. Gesty-Palmer et al.

A fibroblast activation protein α-activatable nanoagent co-delivering diethyldithiocarbamate and copper for tumor therapy and imaging

Disulfiram (DSF), an FDA-approved drug for treating alcoholism, has been verified with Cu2+-dependent anticancer activity by forming Cu(DTC)2, the complex of one of its metabolites diethyldithiocarbamate (DTC) and Cu2+. Nevertheless, the antitumor effect is limited by insufficient Cu(DTC)2 formation in suit and off-target system toxicity. Herein, we developed a fibroblast activation protein α (FAPα) activatable nanoagent (HfD-HID-Cu) for co-delivery of DTC polymeric prodrug and exogenous Cu2+ to achieve enhanced cancer-specific therapy and activatable in situ fluorescence imaging meanwhile. HfD-HID-Cu was simply constructed through the co-assembly of the DTC polymeric prodrug (HA-fap-DTC) and the copper-loaded IR808-conjugated polymer (HA-IR-DPA-Cu), which could serve as the “OFF-to-ON” switch for chemotherapy and fluorescence. With the high expression of FAPα in tumor tissues, HA-fap-DTC could be activated specifically to release DTC, while maintaining inactive in normal tissues. The liberated DTC within tumor tissues could contend for Cu2+ from HA-IR-DPA-Cu, resulting in the formation of highly cytotoxic Cu(DTC)2in situ for chemotherapy, concomitant with the fluorescence recovery of cyanine dye for tumor imaging. This work provides an effective strategy for co-delivery of DTC prodrug and Cu2+ for tumor theranostic with improved selectivity and minimal side effects. Statement of significanceDSF-based antitumor therapy is highly dependent on Cu2+. However, the non-synchronous distribution of DSF/DTC and Cu2+ in tumor tissues attenuates the antitumor efficacy. The insufficient Cu(DTC)2 formation in suit and off-target distribution greatly limit the anti-tumor application. This study provides a nanoagent for co-delivery of DTC polymeric prodrug and Cu2+ by simple co-assembly to achieve their synchronous tumor distribution. It can be selectively activated by FAPα, forming cytotoxic Cu(DTC)2in suit for tumor-specific chemotherapy and reducing the systemic toxicity. In addition to chemotherapy, the nanoagent can emit fluorescence under the sequential triggering of FAPα and released DTC for tumor imaging. Overall, this study renders a promising strategy for improved Cu(DTC)2-based antitumor therapy and imaging.

Phosphorylation patterns in the AT1R C-terminal tail specify distinct downstream signaling pathways.

Different ligands stabilize specific conformations of the angiotensin II type 1 receptor (AT1R) that direct distinct signaling cascades mediated by heterotrimeric G proteins or β-arrestin. These different active conformations are thought to engage distinct intracellular transducers because of differential phosphorylation patterns in the receptor C-terminal tail (the "barcode" hypothesis). Here, we identified the AT1R barcodes for the endogenous agonist AngII, which stimulates both G protein activation and β-arrestin recruitment, and for a synthetic biased agonist that only stimulates β-arrestin recruitment. The endogenous and β-arrestin-biased agonists induced two different ensembles of phosphorylation sites along the C-terminal tail. The phosphorylation of eight serine and threonine residues in the proximal and middle portions of the tail was required for full β-arrestin functionality, whereas phosphorylation of the serine and threonine residues in the distal portion of the tail had little influence on β-arrestin function. Similarly, molecular dynamics simulations showed that the proximal and middle clusters of phosphorylated residues were critical for stable β-arrestin-receptor interactions. These findings demonstrate that ligands that stabilize different receptor conformations induce different phosphorylation clusters in the C-terminal tail as barcodes to evoke distinct receptor-transducer engagement, receptor trafficking, and signaling.

Exploring the value of FAP-targeted PET/CT in differentiating breast cancer molecular subtypes: a preliminary study.

This prospective study aims to evaluate the value of [18F]AlF-NOTA-fibroblast activation protein inhibitor (FAPI)-04 positron emission tomography-computed tomography (PET/CT) in predicting molecular subtypes of breast cancer. The study consecutively recruited patients suspected of having breast cancer from a single center who were prospectively enrolled from July 2023 to May 2024 and underwent [18F]AlF-NOTA-FAPI-04 PET/CT. This study compared the differences in tracer uptake among breast cancers with different adverse prognostic factors and molecular subtypes. The classification performance for each molecular subtype of breast cancer was assessed using a receiver operating characteristic (ROC) curve. Fifty-three participants (mean age, 51 ± 11 years; 52 females) were evaluated. Breast cancer lesions with adverse prognostic factors showed higher tracer uptake. The five different molecular subtypes exhibited varying levels of uptake. The luminal A and luminal B (HER2-negative) subtypes had relatively low uptake, while the luminal B (HER2-positive), HER2-positive, and triple-negative subtypes had relatively high uptake. ROC analysis identified the max standardized uptake value (SUVmax) as a significant classifier (AUC = 0.912, P = 0.0005) for the luminal A subtype, with 100% sensitivity and 83% specificity. For predicting the luminal B (HER2-negative) subtype, SUVmax had an AUC of 0.770 (P = 0.0015). SUVmax, with an AUC of 0.781 (P = 0.003), was used to identify the triple-negative subtype tumors, resulting in a sensitivity of 100% and specificity of 51%. Lastly, the ROC curve showed the cut-off 15.40 (AUC = 0.921, P < 0.0001) could classify luminal A & luminal B (HER2-negative), and luminal B (HER2-positive) & HER2-positive & triple-negative, yielding a sensitivity of 94% and specificity of 79%. The uptake of [18F]AlF-NOTA-FAPI-04 is significantly correlated with the molecular subtypes of breast cancer, and [18F]AlF-NOTA-FAPI-04 PET/CT is a potential tool for noninvasive identification of luminal A subtypes and guidance of FAP-targeted therapies.

Dual targeting chimeric antigen receptor cells enhance antitumour activity by overcoming T cell exhaustion in pancreatic cancer.

Although our previous data indicated that claudin 18 isoform 2 (CLDN18.2)-targeted chimeric antigen receptor(CAR)T cells displayed remarkable clinical efficacy in CLDN18.2-positive gastric cancer, their efficacy is limited in pancreatic ductal adenocarcinoma (PDAC). The tumour microenvironment (TME) is one of the main obstacles to the efficacy of CAR-T and remodelling the TME may be a possible way to overcome this obstacle. The TME of PDAC is characterized by abundant cancer-related fibroblasts (CAFs), which hinder the infiltration and function of CLDN18.2-targeted CAR-T cells. The expression of fibroblast activation protein alpha (FAP) is an important feature of active CAFs, providing potential targets for eliminating CAFs. In this study, we generated 10 FAP/CLDN 18.2 dual-targeted CAR-T cells and evaluated their anti-tumour ability in vitro and in vivo. Compared with conventional CAR-T cells, some dual-targeted CAR-T cells showed improved therapeutic effects in mouse pancreatic cancers. Further, dual-targeted CAR-T cells with better anti-tumour effect could suppress the recruitment of myeloid-derived suppressor cells (MDSCs) to improve the immunosuppressive TME, which contributes to the survival of CD8+ T cells. Moreover, dual-targeted CAR-T cells reduced the exhaustion of T cells in transforming TGF-β dependent manner. The dual-targeted CAR-T cells obtained enhancement of T effector function, inhibition of T cell exhaustion, and improvement of tumour microenvironment. Our findings provide a theoretical rationale for dual-targeted FAP/CLDN 18.2 CAR-T cells therapy in PDAC.

Penilumamide, a novel SIRT1 activator, protects UVB-induced photodamages in HaCaT cells

ABSTRACT Ultraviolet-B (UVB) radiation is a major physical factor that induces structural changes in human skin. The aim of this study was to determine whether the novel silent information regulator 1 (sirtuin 1 SIRT1) protein activator, penilumamide, exerted any protective effects against UVB-induced skin damage using human HaCaT keratinocytes as a model. Enzymatic assays were performed to determine the SIRT1-activating ability of penilumamide, which was compared with that of resveratrol, a potent natural product SIRT1 activator with antioxidant and anti-inflammatory properties. Penilumamide markedly activated SIRT1 enzyme activity compared to resveratrol. To further investigate the protective effect of penilumamide against UVB-induced cytotoxicity, HaCaT cells were pretreated with penilumamide (10 μM) for 24 hr followed by irradiation with UVB (40 mJ/cm2). UVB (40 mJ/cm2) irradiation significantly reduced cell viability in a time-dependent manner, whereas pretreatment with penilumamide blocked this effect. Further, penilumamide decreased the levels of intracellular reactive oxygen species (ROS) generated by UVB irradiation in HaCaT cells. Pretreatment with penilumamide also prevented UVB irradiation-induced changes in mitochondrial membrane potential (ΔΨ m). In addition, pretreatment with penilumamide significantly reduced the expression levels of pro-inflammatory cytokines, interleukin (IL)-6, IL-8, and IL-10 and phosphorylation of nuclear factor-kB (NF-kB). These results indicate that penilumamide protects HaCaT cells from UVB-induced inflammation. Taken together data demonstrate that penilumamide exerted protective effects against UVB-induced ROS generation in HaCaT cells. Therefore, penilumamide may be considered to be used as a new SIRT1 activator to protect human keratinocyte against UVB-induced damage.

Hypermethylated RASAL1’s promotive role in chemoresistance and tumorigenesis of choriocarcinoma was regulated by TET2 but not DNMTs

BackgroundPatients with choriocarcinoma (CC) accompanying chemoresistance conventionally present a poor prognosis. Whether ras protein activator like-1 (RASAL1) functions as a tumor promoter or suppressor depends on tumor types. However, the role of RASAL1 in process of chemoresistance of CC and underlying molecular mechanism remain elusive.MethodsThe expression pattern of RASAL1 in CC cells and tissues was measured using Western blotting, immunohistochemistry and qRT-PCR. Cell viability and proliferative ability were assessed by MTT assay, Tunnel assay and flow cytometric analysis. Additionally, the stemness was evaluated by the colony formation and tumor sphere formation. Methotrexate (MTX) was applied to exam the chemosensitivity of CC cells.ResultsThe expression of RASAL1 was reduced both at the protein and mRNA levels in CC tissues and cells compared to hydatidiform mole (HM) and invasive mole (IM). Loss of RASAL1 was attributed to its promoter hypermethylation and could be restored by 5-Aza. Knock-down of RASAL1 promoted the viability, proliferative potential, stemness and EMT phenotype of JEG-3 cells. However, induced overexpression of RASAL1 by 5-Aza significantly prohibited cell proliferation and stemness potential of the JAR cell. Additionally, the xenograft model indicated that knockdown of RASAL1 led to a remarkable increase of tumor volume and weight in comparison with its counterpart. Moreover, the stimulatory activity brought by decrease of RASAL1 could be deprived by β-catenin inhibitor XAV 939, yet the suppressive activity resulted from its promoter demethylation could be rescued by β-catenin activator BML-284, indicating that function of RASAL1 depends on β-catenin. Besides, the co-immunoprecipitation assay confirmed the physical binding between RASAL1 and β-catenin. Further investigations showed hypermethylated RASAL1 was regulated by TET2 but not DNMTs.ConclusionTaken together, the present data elucidated that reduced RASAL1 through its promoter hypermethylation regulated by TET2 promoted the tumorigenicity and chemoresistance of CC via modulating β-catenin both in vitro and in vivo.

Iron regulatory protein 1 is required for the propagation of inflammation in inflammatory bowel disease

Inflammatory bowel diseases (IBD) are complex disorders. Iron accumulates in the inflamed tissue of IBD patients, yet neither a mechanism for the accumulation nor its implication on the course of inflammation are known. We hypothesized that the inflammation modifies iron homeostasis, affects tissue iron distribution and that this in turn perpetuates the inflammation. This study analyzed human biopsies, animal models and cellular systems to decipher the role of iron homeostasis in IBD. We found inflammation-mediated modifications of iron distribution, and iron-decoupled activation of the iron regulatory protein (IRP)1. To understand the role of IRP1 in the course of this inflammation-associated iron pattern, a novel cellular co-culture model was established, that replicated the iron-pattern observed in vivo, and supported involvement of nitric oxide in the activation of IRP1 and the typical iron pattern in inflammation. Importantly, deletion of IRP1 from an IBD mouse model completely abolished both, the misdistribution of iron and intestinal inflammation. These findings suggest that IRP1 plays a central role in the coordination of the inflammatory response in the intestinal mucosa and that it is a viable candidate for therapeutic intervention in IBD.

A comparative review of the application value of FAPI PET/CT and 18F-FDG PET/CT in lung cancer.

Fibroblast activation protein inhibitor (FAPI) positron emission tomography/computed tomography (PET/CT) is a multimodal imaging technique that combines PET and CT, utilizing FAP inhibitors as radiotracers. Fibroblast activation protein, a serine protease highly expressed in many epithelial tumor-associated fibroblasts, plays a crucial role in tumor stroma formation and remodeling. Through the detection of FAP expression, FAPI PET/CT facilitates the diagnosis and staging of both benign and malignant pulmonary tumors. In contrast to traditional fluorine-18-fluorodeoxyglucose (18F-FDG) PET/CT focusing on glucose metabolism, FAPI PET/CT offers benefits such as enhanced specificity, reduced background noise, accelerated imaging speed, and decreased radiation exposure. This review provides an overview of the progress in applying FAPI PET/CT and 18F-FDG PET/CT in pulmonary malignancies and discusses current challenges and future prospects.

Initial clinical experience using 68Ga-FAPI-46 PET/CT for detecting various cancer types.

Numerous studies have shown that gallium-68-labeled fibroblast activation protein inhibitor (68Ga-FAPI) positron emission tomography/computed tomography (PET/CT) scans would yield high intra-tumoral tracer uptake and low uptake in normal tissues as background, thus allowing for excellent visualization of lesions in the cancer microenvironment. This study set out to compare the suitability of novel 68Ga-FAPI-46 PET versus routine fluorine-18-fluorodeoxyglucose (18F-FDG) PET and other few cases of 68Ga-DOTATATE/68Ga-Pentixafor PET/CT for the assessment of different types of cancer. A retrospective analysis of 11 patients (6 males, 5 females; average age: 53 years, range: 10-58 years) with histopathologically confirmed, well-differentiated adenocarcinoma, medullar thyroid cancer (MTC), papillary thyroid carcinoma (PTC), cervical, gastric, glioblastoma multiform (GBM), colon, Ewing's sarcoma, and breast cancer was performed. These patients underwent PET/CT scans using four different radiotracers (9 18F-FDG, 11 68Ga- FAPI, 3 68Ga-DOTATATE, and 1 68Ga-Pentixafor). The patients' PET/CT images were visually evaluated for cancer detection, and analyzed semi-quantitatively through image- derived metrics, such as target-to-background ratio (TBR) and maximum standardized uptake value (SUVmax), for recurrence and metastasis. The study of 11 patients revealed that 68Ga-FAPI-46 was more effective than other tracers for detecting metastases, with 55 vs. 49 metastases in the lymph nodes, 4 vs. 3 in the liver, and 4 vs. 3 in the bones detected in comparison to 18F-FDG. No significant differences were observed in 68Ga-DOTATATE and 68Ga-Pentixafor PET images. In addition, in five patients, the SUVmax and TBR values in 68Ga-FAPI-46 PET images were significantly higher than those in 18F-FDG PET images for lymph nodes and bone metastases. Although the SUVmax in 68Ga-FAPI-46 and 18F-FDG PET images for liver metastases was comparable, 68Ga-FAPI- 46 had a significantly higher TBR than 18F-FDG. Our findings suggest that FAPI PET/CT is not suitable for evaluating GBM and Ewing sarcoma but generally outperforms 18F-FDG PET/CT in various types of breast cancer, gastrointestinal, gynecological, PTC and MTC. However, larger trials are needed to validate these preliminary findings.

Localized in vivo prodrug activation using radionuclides.

We screened the ability of radionuclides to chemically activate a model radiation-activated prodrug consisting of the microtubule destabilizing monomethyl auristatin E caged by a radiation-responsive phenyl azide ("caged-MMAE") and interpreted experimental results using the radiobiology computational simulation suite TOPAS-nBio. RAiDER was evaluated in syngeneic mouse models of cancer using fibroblast activation protein inhibitor (FAPI) agents 99m Tc-FAPI-34 and 177 Lu-FAPI-04, the prostate-specific membrane antigen (PSMA) agent 177 Lu-PSMA-617, combined with caged-MMAE or caged-exatecan. Biodistribution in mice, combined with clinical dosimetry, estimated the relationship between radiopharmaceutical uptake in patients and anticipated concentrations of activated prodrug using RAiDER. RAiDER efficiency varied by 250-fold across radionuclides ( 99m Tc> 177 Lu> 64 Cu> 68 Ga> 223 Ra> 18 F), yielding up to 1.22µM prodrug activation per Gy of exposure from 99m Tc. Computational simulations implicated low-energy electron-mediated free radical formation as driving prodrug activation. Clinically relevant radionuclide concentrations chemically activated caged-MMAE restored its ability to destabilize microtubules and increased its cytotoxicity by up to 600-fold compared to non-irradiated prodrug. Mice treated with 99m Tc-FAPI-34 and caged-MMAE accumulated up to 3000× greater concentrations of activated MMAE in tumors compared to other tissues. RAiDER with 99m Tc-FAPI-34 or 177 Lu-FAPI-04 delayed tumor growth, while monotherapies did not ( P <0.03). Clinically-guided dosimetry suggests sufficient radiation doses can be delivered to activate therapeutically meaningful levels of prodrug. This proof-of-concept study shows that RAiDER is compatible with multiple radionuclides commonly used in nuclear medicine and has the potential to improve the efficacy of radiopharmaceutical therapies to treat cancer safely. RAiDER thus shows promise as an effective strategy to treat disseminated malignancies and broadens the capability of radiopharmaceuticals to trigger diverse biological and therapeutic responses.

Inositol 1,4,5-Trisphosphate Receptors Regulate Vascular Smooth Muscle Cell Proliferation and Neointima Formation in Mice.

Vascular smooth muscle cell (VSMC) proliferation is involved in many types of arterial diseases, including neointima hyperplasia, in which Ca2+ has been recognized as a key player. However, the physiological role of Ca2+ release via inositol 1,4,5-trisphosphate receptors (IP3Rs) from endoplasmic reticulum in regulating VSMC proliferation has not been well determined. Both invitro cell culture models and invivo mouse models were generated to investigate the role of IP3Rs in regulating VSMC proliferation. Expression of all 3 IP3R subtypes was increased in cultured VSMCs upon platelet-derived growth factor-BB and FBS stimulation as well as in the left carotid artery undergoing intimal thickening after vascular occlusion. Genetic ablation of all 3 IP3R subtypes abolished endoplasmic reticulum Ca2+ release in cultured VSMCs, significantly reduced cell proliferation induced by platelet-derived growth factor-BB and FBS stimulation, and also decreased cell migration of VSMCs. Furthermore, smooth muscle-specific deletion of all IP3R subtypes in adult mice dramatically attenuated neointima formation induced by left carotid artery ligation, accompanied by significant decreases in cell proliferation and matrix metalloproteinase-9 expression in injured vessels. Mechanistically, IP3R-mediated Ca2+ release may activate cAMP response element-binding protein, a key player in controlling VSMC proliferation, via Ca2+/calmodulin-dependent protein kinase II and Akt. Loss of IP3Rs suppressed cAMP response element-binding protein phosphorylation at Ser133 in both cultured VSMCs and injured vessels, whereas application of Ca2+ permeable ionophore, ionomycin, can reverse cAMP response element-binding protein phosphorylation in IP3R triple knockout VSMCs. Our results demonstrated an essential role of IP3R-mediated Ca2+ release from endoplasmic reticulum in regulating cAMP response element-binding protein activation, VSMC proliferation, and neointima formation in mouse arteries.

DNA mismatch repair defect and intratumor heterogeneous deficiency differently impact immune responses in diffuse large B-cell lymphoma

ABSTRACT Deficient (d) DNA mismatch repair (MMR) is a biomarker predictive of better response to PD-1 blockade immunotherapy in solid tumors. dMMR can be caused by mutations in MMR genes or by protein inactivation, which can be detected by sequencing and immunohistochemistry, respectively. To investigate the role of dMMR in diffuse large B-cell lymphoma (DLBCL), MMR gene mutations and expression of MSH6, MSH2, MLH1, and PMS2 proteins were evaluated by targeted next-generation sequencing and immunohistochemistry in a large cohort of DLBCL patients treated with standard chemoimmunotherapy, and correlated with the tumor immune microenvironment characteristics quantified by fluorescent multiplex immunohistochemistry and gene-expression profiling. The results showed that genetic dMMR was infrequent in DLBCL and was significantly associated with increased cancer gene mutations and favorable immune microenvironment, but not prognostic impact. Phenotypic dMMR was also infrequent, and MMR proteins were commonly expressed in DLBCL. However, intratumor heterogeneity existed, and increased DLBCL cells with phenotypic dMMR correlated with significantly increased T cells and PD-1+ T cells, higher average nearest neighbor distance between T cells and PAX5+ cells, upregulated immune gene signatures, LE4 and LE7 ecotypes and their underlying Ecotyper-defined cell states, suggesting the possibility that increased T cells targeted only tumor cell subsets with dMMR. Only in patients with MYC¯ DLBCL, high MSH6/PMS2 expression showed significant adverse prognostic effects. This study shows the immunologic and prognostic effects of genetic/phenotypic dMMR in DLBCL, and raises a question on whether DLBCL-infiltrating PD-1+ T cells target only tumor subclones, relevant for the efficacy of PD-1 blockade immunotherapy in DLBCL.

A neurodevelopmental disorder mutation locks G proteins in the transitory pre-activated state

Many neurotransmitter receptors activate G proteins through exchange of GDP for GTP. The intermediate nucleotide-free state has eluded characterization, due largely to its inherent instability. Here we characterize a G protein variant associated with a rare neurological disorder in humans. GαoK46E has a charge reversal that clashes with the phosphate groups of GDP and GTP. As anticipated, the purified protein binds poorly to guanine nucleotides yet retains wild-type affinity for G protein βγ subunits. In cells with physiological concentrations of nucleotide, GαoK46E forms a stable complex with receptors and Gβγ, impeding effector activation. Further, we demonstrate that the mutant can be easily purified in complex with dopamine-bound D2 receptors, and use cryo-electron microscopy to determine the structure, including both domains of Gαo, without nucleotide or stabilizing nanobodies. These findings reveal the molecular basis for the first committed step of G protein activation, establish a mechanistic basis for a neurological disorder, provide a simplified strategy to determine receptor-G protein structures, and a method to detect high affinity agonist binding in cells.