Active TGF-beta Research Articles

A-118 Targeting TGF-beta activation in cutaneous T-cell lymphomas

A-118 Targeting TGF-beta activation in cutaneous T-cell lymphomas

Five latent factors underlie response to immunotherapy

Only a subset of patients treated with immune checkpoint inhibitors (CPIs) respond to the treatment, and distinguishing responders from non-responders is a major challenge. Many proposed biomarkers of CPI response and survival probably represent alternative measurements of the same aspects of the tumor, its microenvironment or the host. Thus, we currently ignore how many truly independent biomarkers there are. With an unbiased analysis of genomics, transcriptomics and clinical data of a cohort of patients with metastatic tumors (n = 479), we discovered five orthogonal latent factors: tumor mutation burden, T cell effective infiltration, transforming growth factor-beta activity in the microenvironment, prior treatment and tumor proliferative potential. Their association with CPI response and survival was observed across all tumor types and validated across six independent cohorts (n = 1,491). These five latent factors constitute a frame of reference to organize current and future knowledge on biomarkers of CPI response and survival.

The CALR mutations enhance the expression of the immunosuppressive proteins GARP and LAP on peripheral blood lymphocytes through increased binding of activated platelets.

Recently, an antibody which inhibits the glycoprotein A repetitions predominant (GARP)-mediated release of active transforming growth factor beta (TGFβ) from the TGFβ propeptide latency-associated peptide (LAP) showed preclinical activity in a murine model of the chronic myeloproliferative neoplasms (MPN). Consequently, we investigated the expression of the immunosuppressive molecules LAP and GARP on peripheral blood lymphocytes from 56 MPN patients and 11 healthy donors (HD). We found that lymphocytes from patients with MPN express higher levels of LAP and GARP with no strong differences found between the different MPN diagnoses. The impact of clinical parameters on the expression of LAP and GARP by lymphocytes showed that patients with calreticulin (CALR)mut MPN have increased expression compared with HD and patients with the Januskinase2 (JAK2) mutation. The fraction of lymphocytes bound to activated platelets (aPLT) strongly correlate to LAP and GARP expression suggesting that it is not the lymphocytes themselves but aPLT, which confer the increased expression of GARP and LAP on MPN patient lymphocytes. Notably, no differences in neither platelet counts nor anti-thrombotic therapy was identified between patients with JAK2- and CALRmut patients. Analysis of platelet gene expression failed to identify differences in expression of relevant genes between JAK2- and CALRmut patients.

Abstract PO4-22-02: pSmad2 as a marker of radiotherapy benefit in breast cancer- results from the randomized SweBCG91RT trial

Abstract Background: The TGF-beta pathway can have both pro- and antitumoral effects in breast cancer. Experimental studies further suggest that TGF-beta can mediate radioresistance, either through direct effects on tumor cells or indirectly by its immunomodulatory properties. However, the effect of TGF-beta signaling is highly depending on tumor stage, mutational status and microenvironmental cell composition. In order to explore TGF-beta signaling as a potential biomarker or therapeutical target it is therefore important to perform analyses in well-defined study populations. The purpose of this study was to study the effect of active TGF beta signaling in tumor cells, on the risk of ipsilateral breast tumor recurrence (IBTR) and benefit from radiotherapy (RT) in specifically early-stage breast cancer using nuclear, phosphorylated Smad2 (pSmad2) as a marker. Methods: The SweBCG91RT cohort included 1179 patients with stage I-IIA tumors randomized to breast-conserving surgery (BCS) with or without postoperative RT. Nuclear pSmad2 expression was assessed as for 987 available tumors on TMAs as the mean proportion of tumor cells with positive nuclear staining. Based on the distributions, tumors were categorized as having high (≥80%, n=344), medium (21-79%, n=428) or low (≤20%, n=161) pSmad2 staining. The primary endpoint was time to ipsilateral breast tumor recurrence as the first event within 10 years. Results: pSmad2Low tumors were more likely to be of grade III (p< 0.001) and tended to be larger than pSmad2Medium (p=0.002, median 15 vs 12 mm) and pSmad2High (p< 0.001, median 15 vs 12 mm) tumors. No associations between pSmad2 and subtype or age were found. Numerically, tumors with pSmad2low (HR 1.55, CI 95% 0.80-3.02, p=0.19) or pSmad2Medium (HR 2.53, CI 95% 1.52-4.22, p< 0.001) had a higher risk of IBTR than pSmad2high tumors (reference). These findings remained significant after adjustment for age, histological grade, and RT. The benefit from RT was similar for pSmad2High (HR0.41, CI 95% 0.15-1.07, p=0.068), pSmad2Medium (HR 0.36, CI 95% 0.21-0.63, p< 0.001), and pSmad2Low (HR 0.51, CI 95% 0.18-1.46, p=0.21) tumors (pinteraction=0.86). Conclusions: Early-stage breast tumors with high nuclear pSmad2 staining may be at a reduced risk of an IBTR. However, the relative benefit from RT does not seem to differ from tumors with medium or low pSmad2 staining. These results need to be confirmed in additional studies, but they provide initial evidence that tumoral TGF-beta signaling has rather tumor restraining functions in early-stage breast cancer. Next analytical steps should include the tumor mutation status (particularly for TGF-beta signaling components TGFBR2, SMAD4 and TP53), as well as the TGF-beta signaling status within the tumor microenvironment. Citation Format: Per Karlsson, Viktoria Thurfjell, Anikó Kovács, Fredrika Killander, Emma Niméus, Erik Holmberg, Carina Strell, Axel Stenmark Tullberg. pSmad2 as a marker of radiotherapy benefit in breast cancer- results from the randomized SweBCG91RT trial [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-22-02.

Pleiotropic Action of TGF-Beta in Physiological and Pathological Liver Conditions.

The aim of this study is to review and analyze the pleiotropic effects of TGF-β in physiological and pathological conditions of the liver, with particular emphasis on its role in immune suppression, wound healing, regulation of cell growth and differentiation, and liver cell apoptosis. A literature review was conducted, including 52 studies, comprising review articles, in vitro and in vivo studies, and meta-analyses. Only studies published in peer-reviewed scientific journals were included in the analysis. TGF-β is a pleiotropic growth factor that is crucial for the liver, both in physiology and pathophysiology. Although its functions are complex and diverse, TGF-β plays a constant role in immune suppression, wound healing, and the regulation of cell growth and differentiation. In concentrations exceeding the norm, it can induce the apoptosis of liver cells. Increased TGF-β levels are observed in many liver diseases, such as fibrosis, inflammation, and steatosis. TGF-β has been shown to play a key role in many physiological and pathological processes of the liver, and its concentration may be a potential diagnostic and prognostic marker in liver diseases.

Abstract 5458: Plasticity of CAF-like states in Ewing sarcoma is mediated by TGF-beta

Abstract Transcriptional plasticity of cancer cells promotes tumor progression and treatment resistance via generation of phenotypically distinct cell states. We previously showed that Ewing sarcoma (EwS) cells exhibit inter- and intra-tumoral transcriptional heterogeneity and that they undergo state changes in response to cues from the tumor microenvironment (TME). More recently, we identified phenotypically distinct EwS tumor cell subpopulations in vivo that deposit pro-tumorigenic extracellular matrix (ECM) proteins, adopting similar functions to cancer-associated fibroblasts (CAFs). Here, we sought to identify the upstream regulators of these CAF-like EwS cells with the goal of defining molecular mechanisms that control EwS plasticity and creation of ECM-remodeling states. Integration of single cell proteogenomic sequencing (CITE-seq) with immunofluorescence and digital spatial profiling of cell line xenografts and patient tumors validated the cell-surface protein CD73 (NT5E) as a biomarker of ECM-secreting EwS cell subpopulations in vitro and in vivo. The transcriptomes of CD73+ CAF-like cells were compared to isogenic CD73- cells (n=9 cell lines) and highly significant enrichment of gene ontology terms related to TGF-beta signaling (p.adj = <4.4x10-14) was noted. EwS cells were previously reported to be non-responsive to TGF-beta due to profound transcriptional repression of the TGF-beta type 2 receptor (TGFBR2) by EWS::FLI1. In support of this, TGFBR2 expression was low in bulk measurements of EwS cells. However, TGFBR2 mRNA and surface protein expression were significantly increased in CD73+ subpopulations. Exposure of EwS cells to TGF-beta ligands (TGFB1 or TGFB2) strongly induced the CAF-like gene signature, including ECM genes BGN, COL1A1, SPARC, and TNC (p. adj < 8.5x10-5), and this was augmented in CD73+ cell populations. Transduction of EwS cells with dominant negative-TGFBR2 inhibited both basal and TGF-beta-induced expression of CAF-like signature genes and blocked SMAD2 phosphorylation. To test the functional significance of the TGF-beta response, unsorted EwS cells were plated in 2D or as tumor spheroids in collagen I-rich 3D cultures and exposed to TGFB1 or SB-505124, a TGF-beta inhibitor. TGFB1 treatment had no effect on cell viability or proliferation in 2D but increased invasion into collagen in 3D. Inhibition of TGF-beta reduced CAF-like gene expression in 2D cultures and diminished both viability and invasion in 3D. Finally, our studies revealed that NT5E (CD73) is a TGF-beta induced target in EwS cells and that CD73+ cells upregulate expression of TGFB2. Thus, an autocrine signaling circuit is created in which TGF-beta activity in CAF-like tumor cell subpopulations is sustained by CD73+ tumor cell-derived ligand. Together these studies demonstrate that TGF-beta is a key determinant of cell plasticity and ECM remodeling in EwS. Citation Format: Emma Wrenn, April A. Apfelbaum, Katherine Braun, Erin R. Rudzinski, Xuemei Deng, Aya Miyaki, Trisha Lipson, Nicolas M. Garcia, Virginia J. Hoglund, Elizabeth R. Lawlor. Plasticity of CAF-like states in Ewing sarcoma is mediated by TGF-beta [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5458.

Induced Attenuation of Scleral TGF-β Signaling in Mutant Mice Increases Susceptibility to IOP-Induced Optic Nerve Damage.

Axonal optic nerve (ON) damage in glaucoma is characteristically associated with increased amounts of active transforming growth factor-beta 2 (TGF-β2) in the ON head. Here we investigated the functional role of scleral TGF-β signaling in glaucoma. A deficiency of Tgfbr2, which encodes for TGF-β receptor type II (TGF-βRII), the essential receptor for canonical TGF-β signaling, was induced in fibroblasts (including those of the sclera) of mutant mice. To this end, 5-week-old mice were treated with tamoxifen eye drops. Experimental glaucoma was induced in 8-week-old mice using a magnetic microbead (MB) model. After 6 weeks of high intraocular pressure (IOP), the ON axons and their somata in the retina were labeled by paraphenylenediamine (PPD) and RNA-binding protein with multiple splicing (RBPMS) immunohistochemistry, respectively, and quantified. Tamoxifen treatment resulted in a significant decrease of TGF-βRII and its mRNA in the sclera. After 6 weeks of high IOP, reduced numbers of PPD-stained ON axons were seen in MB-injected eyes in comparison with not-injected contralateral eyes. Moreover, MB injection also led to a decrease of retinal ganglion cell (RGC) somata as seen in RBPMS-stained retinal wholemounts. Axon loss and RGC loss were significantly higher in mice with a fibroblast specific deficiency of TGF-βRII in comparison with control animals. We conclude that the ablation of scleral TGF-β signaling increases the susceptibility to IOP-induced ON damage. Scleral TGF-β signaling in mutant mice appears to be beneficial for ON axon survival in experimentally induced glaucoma.

Abstract 15735: Antibody-Based Plasma Proteomics Profiling Reveals New Markers of Cardiorespiratory Fitness

Background: Plasma proteins can be biomarkers of cardiovascular health status but also physiologic effectors that mediate health benefits. Cardiorespiratory fitness (CRF) is an integrative measure of cardiovascular and metabolic health and independent predictor of future cardiovascular disease (CVD) and mortality risk, however limited knowledge exists regarding its molecular transducers. We sought to expand upon prior proteomic studies of CRF using an antibody-based technology (Olink TM ). Hypothesis: Olink proteomic profiling will identify new markers and potential mediators of CRF. Methods: We measured plasma proteins (N=1,472) using Olink’s platform in a pilot study within the HERITAGE Family Study (N=209 participants, mean age=34 years, 56% female, 36% Black) before and after 20 weeks of endurance exercise training (ET). We performed multivariable linear regression to measure the association between protein levels and CRF measured by CPET (VO 2 max in ml*kg -1 *min -1 ) adjusting for age and sex. Lean body mass was measured by hydrostatic weighing. Protein changes after ET were assessed using paired Student’s t-tests. Results: We identified 70 proteins significantly associated (FDR q <0.05) with VO 2 max. Among these, 14 have not previously been measured in the context of CRF, including perilipin-1, a modulator of lipid homeostasis in adipocytes (beta=-3.8, q=2.4x10 -4 ); latent-transforming growth factor beta-binding protein 3 (beta=-2.6, q=1.3x10 -3 ), a key regulator of TGF-beta activation; and hydroxysteroid 11-beta dehydrogenase 1 (beta=3.2, q=1.2x10 -3 ), which regulates cortisol metabolism. Carbonic anhydrase (CA) XIV (CA14; beta=5.4, q=1.3x10 -8 ), a member of the CA family with extracellular activity, had the strongest positive association with VO 2 max in our primary model as well as after further adjusting VO 2 max for lean body mass. Further, CA14 levels increased significantly after ET (log2 fold change: 0.14, q=0.007). Conclusions: Antibody-based plasma proteomics profiling identified new markers of CRF, including a CA isoform that increases after ET. These findings motivate further study of CA14’s mechanistic role in CA14 and expanded proteomics profiling to investigate molecular transducers of CRF.

Molecular mechanisms controlling the biogenesis of the TGF-β signal Vg1

The TGF-beta signals Vg1 (Dvr1/Gdf3) and Nodal form heterodimers to induce vertebrate mesendoderm. The Vg1 proprotein is a monomer retained in the endoplasmic reticulum (ER) and is processed and secreted upon heterodimerization with Nodal, but the mechanisms underlying Vg1 biogenesis are largely elusive. Here, we clarify the mechanisms underlying Vg1 retention, processing, secretion, and signaling and introduce a Synthetic Processing (SynPro) system that enables the programmed cleavage of ER-resident and extracellular proteins. First, we find that Vg1 can be processed by intra- or extracellular proteases. Second, Vg1 can be processed without Nodal but requires Nodal for secretion and signaling. Third, Vg1-Nodal signaling activity requires Vg1 processing, whereas Nodal can remain unprocessed. Fourth, Vg1 employs exposed cysteines, glycosylated asparagines, and BiP chaperone-binding motifs for monomer retention in the ER. These observations suggest two mechanisms for rapid mesendoderm induction: Chaperone-binding motifs help store Vg1 as an inactive but ready-to-heterodimerize monomer in the ER, and the flexibility of Vg1 processing location allows efficient generation of active heterodimers both intra- and extracellularly. These results establish SynPro as an in vivo processing system and define molecular mechanisms and motifs that facilitate the generation of active TGF-beta heterodimers.

Overcoming the Challenges of Phytochemicals in Triple Negative Breast Cancer Therapy: The Path Forward.

Triple negative breast cancer (TNBC) is a very aggressive subtype of breast cancer that lacks estrogen, progesterone, and HER2 receptor expression. TNBC is thought to be produced by Wnt, Notch, TGF-beta, and VEGF pathway activation, which leads to cell invasion and metastasis. To address this, the use of phytochemicals as a therapeutic option for TNBC has been researched. Plants contain natural compounds known as phytochemicals. Curcumin, resveratrol, and EGCG are phytochemicals that have been found to inhibit the pathways that cause TNBC, but their limited bioavailability and lack of clinical evidence for their use as single therapies pose challenges to the use of these phytochemical therapies. More research is required to better understand the role of phytochemicals in TNBC therapy, or to advance the development of more effective delivery mechanisms for these phytochemicals to the site where they are required. This review will discuss the promise shown by phytochemicals as a treatment option for TNBC.

Abstract B049: Radiogenomic profiling of prostate tumors prior to external beam radiotherapy (EBRT) converges on a transcriptomic signature of TGF-beta activity driving tumor recurrence

Abstract Background: In patients who receive definitive therapy for locally advanced prostate cancer, biochemical recurrence is due to local recurrence and/or the presence of undetected metastatic disease at the time of initial diagnosis and treatment. Approximately 25-40% of patients who receive radiotherapy plus androgen deprivation therapy (ADT) with curative intent are expected to recur within 5-10 years. We hypothesized that applying multiparametric (mp) MRI, gene expression, and genomic analyses to prostate tumors prior to EBRT+ADT would identify biomarkers predictive of recurrence (NCT01834001). Methods: At baseline and 6 months after completion of radiotherapy, mpMRI was performed and lesions were manually contoured. MR/ultrasound-fusion biopsies acquired at baseline targeted to up to three distinct MR lesions per patient. 29 patients with intermediate and high risk localized prostate cancer treated with EBRT+ADT with sufficient baseline biopsy tissue were selected for this analysis (median follow-up: 91 months). Tumor tissues were macrodissected to obtain DNA and RNA from 60 distinct biopsy regions (1-4 per patient). DNA was used for generating whole-exome sequencing libraries (92.5 × median coverage); RNA was converted into cDNA and hybridized to Affymetrix Human Exon 1.0 microarrays. Secondary gene expression cohorts with clinical annotation were obtained from the University of Miami and Queen’s University Belfast. Gene expression signatures were processed using Ingenuity Pathway Analysis (IPA) and the Decipher GRID. Results: Five of the 29 patients experienced biochemical failure by the time of data analysis; four of these patients were identified to have distant metastases with no evidence of local recurrence at the time of biochemical failure. Baseline and posttreatment lesion volumes by MRI and posttreatment ADCmax (apparent diffusion coefficient) were positively associated with recurrence (p<0.05). The Decipher signature positively correlated with these three MR features. Comparison of gene expression using a linear mixed-effect model identified 1,120 differentially expressed transcripts. IPA nominated TGF-beta signaling as the topmost enriched upstream regulator (z=3.32). This result was validated in the Miami (z=2.54) and Belfast (z=3.67) cohorts. By network analysis, genes that independently tracked with the baseline and posttreatment volumes and posttreatment ADCmax MR features similarly converged to the TGF-beta pathway. Co-occurrence of biallelic TP53 alterations with 1- or 2-copy PTEN losses was observed exclusively in patients who recurred. Conclusions: Genomic, transcriptomic and radiomic analyses have nominated predictive biomarkers that identify the subset of patients with locally advanced prostate cancer destined to recur after definitive EBRT+ADT. Identification of molecular features predictive of failure suggests that aggressive pre-existing subclones harboring these alterations have metastatic potential. Emerging systemic therapies including TGF-beta ligand traps targeted to these tumors may improve overall outcomes. Citation Format: Adam G. Sowalsky, Anson T. Ku, Uma Shankavaram, Shana Y. Trostel, Houssein A. Sater, Stephanie A. Harmon, Nicole V. Carrabba, Yang Liu, Radka Stoyanova, Elai Davicioni, Alan Pollack, Baris Turkbey, Deborah E. Citrin. Radiogenomic profiling of prostate tumors prior to external beam radiotherapy (EBRT) converges on a transcriptomic signature of TGF-beta activity driving tumor recurrence [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B049.

Abstract 5198: Humanized mice for preclinical efficacy evaluation of drugs targeting GARP

Abstract Introduction: GARP (Glycoprotein-A repetitions predominant) is a cell surface receptor which is coded by Lrrc32 (Leucine rich repeat containing protein 32) and involved in the activation of TGF-beta. The expression of GARP has been widely found in various cells including Treg, activated B cells, mesenchymal stromal cells and many types of cancer cells. In the tumor microenvironment (TME), GARP from Treg promotes the growth of tumor via secretion of immune suppressive TGF-beta. Result: We established GARP humanized mouse model (hGARP) on C57BL/6 background in which the signal peptide and extracellular domain of mouse GARP (mGARP) was replaced by human GARP (hGARP). Similar expression level of hGARP on Treg cells in splenocytes from C57BL/6-hGARP mice was observed compared with mGARP in wildtype mice. After treating with anti-hGARP antibody, tumor growth was inhibited in the cohort engrafted with MC38 colorectal cancer cell line. Flow cytometry analysis of the tumor infiltrating lymphocytes revealed an abundant accumulation of hGARP on the Treg cells suggesting that blocking of hGARP in Treg cells contributed to the therapeutic efficiency. In addition, hGARP expression was also found on platelet cells from C57BL/6-hGARP mice, which is similar to human. After treating with anti-hGARP, we also found a significant reduction of platelet cells in C57BL/6-hGARP mice. In vivo assay suggested that the reduction of platelet cells from C57BL/6-hGARP mice correlated well with the binding of anti-hGARP. Conclusion: C57BL/6-hGARP is a well-established mouse model for the pre-clinical evaluation of both therapeutic effect and toxicity of anti-hGARP therapy. Citation Format: Xing Liu, Meirong Wu, Huiyi Wang, Weiwei Yu, Jianming Xu, Hongyan Sun, Cunxiang Ju, Hongyu Wang, Santi Suryani Chen, Zhiying Li, Mark Wade Moore, Jing Zhao, Xiang Gao. Humanized mice for preclinical efficacy evaluation of drugs targeting GARP. [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 5198.

Long-term platinum-based drug accumulation in cancer-associated fibroblasts promotes colorectal cancer progression and resistance to therapy

A substantial proportion of cancer patients do not benefit from platinum-based chemotherapy (CT) due to the emergence of drug resistance. Here, we apply elemental imaging to the mapping of CT biodistribution after therapy in residual colorectal cancer and achieve a comprehensive analysis of the genetic program induced by oxaliplatin-based CT in the tumor microenvironment. We show that oxaliplatin is largely retained by cancer-associated fibroblasts (CAFs) long time after the treatment ceased. We determine that CT accumulation in CAFs intensifies TGF-beta activity, leading to the production of multiple factors enhancing cancer aggressiveness. We establish periostin as a stromal marker of chemotherapeutic activity intrinsically upregulated in consensus molecular subtype 4 (CMS4) tumors and highly expressed before and/or after treatment in patients unresponsive to therapy. Collectively, our study underscores the ability of CT-retaining CAFs to support cancer progression and resistance to treatment.

Abstract C039: An EGFR-activated subset of myofibroblastic cancer-associated fibroblasts supports local metastatic dissemination in pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDA) has a dismal prognosis and new therapies are needed. Cancer-associated fibroblasts (CAFs) can promote PDA progression and are recognized promising therapeutic targets. However, the ongoing characterization of PDA CAF heterogeneity and plasticity and the identification of potential tumor-restraining CAF populations have highlighted the need to further dissect the diverse biology of PDA CAFs to develop effective combination therapies. Using pancreatic organoid/fibroblast co-cultures and organoid-derived transplantation mouse models of PDA, we mechanistically investigated the signaling cascade downstream TGF-beta activation, which is a main driver of myofibroblastic CAF (myCAF) formation. We find that the EGFR/ERBB2 pathway is activated by TGF-beta in a subset of myCAFs, in both murine and human PDA, via amphiregulin induction and autocrine signaling. ERBB activation is important for the maintenance of this myCAF subset, with genetic and pharmacological inhibition of this pathway differentially affecting PDA CAF composition both in culture and mouse models. Importantly, analysis of lung cancer and breast cancer patient samples suggests that our observations in PDA are also relevant to these malignancies. Finally, we reveal a role of this EGFR-activated myCAF subset in promoting local metastatic dissemination in PDA and indicate new potential combination strategies. Our study highlights the need to better dissect PDA CAF heterogeneity also in populations typically considered to be tumor-restraining, in order to develop effective combination strategies for future clinical intervention. Our work also reveals how cancer-targeting therapies can directly affect the surrounding microenvironment with potential clinical implications for various malignancies. Citation Format: Gianluca Mucciolo, Joaquín Araos Henríquez, Sara Pinto Teles, Judhell Manansala, Wenlong Li, Eloise G. Lloyd, Priscilla Cheng, Giulia Biffi. An EGFR-activated subset of myofibroblastic cancer-associated fibroblasts supports local metastatic dissemination in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C039.

Avb6 Integrin-Targeted PET Imaging to Monitor Radiation-Induced Pulmonary Fibrosis In Vivo

<h3>Purpose/Objective(s)</h3> Radiation-induced pulmonary fibrosis (RIPF) is a common, debilitating late complication from radiation therapy, with poor prognosis and limited treatment options. RIPF is a particularly challenging dose-limiting normal tissue toxicity in pediatric hematological malignancies after total body irradiation, lung cancers, breast cancers, and thoracic lymphomas. Currently, clinical monitoring of RIPF progression remains challenging given the lack of imaging tools that can reliably detect RIPF at an early stage and the difficulty distinguishing RIPF from tumor recurrence. In the lung, α_vβ₆ integrin plays a central role in the pathogenesis of RIPF, by serving as a major TGF-beta activator after radiation injury. The objective of this study is to investigate the use of α_vβ₆ integrin-targeted PET imaging (using ⁶⁸Ga-A20FMDV2) to monitor radiation-induced pulmonary fibrosis in a murine model. <h3>Materials/Methods</h3> Using the small animal radiation research platform, the right lungs of C57BL/6J female mice (aged 8-10 weeks) were irradiated focally at 70 Gy using a 3 × 3 mm beam collimator, while the contralateral lung served as control. In addition, sham-treated animals served as control. This high dose focal irradiation model was used as prior studies demonstrated fibrosis in this model starting at 4-6 weeks post-RT. At 6 weeks post-RT, animals were administered 7.4 MBq of ⁶⁸Ga-A20FMDV2 through IV injection and imaged 1 hour later using a small animal micro-PET/CT system. Micro-PET/CT fusion images were reconstructed and maximum intensity projection images were generated. <h3>Results</h3> At 6 weeks post-RT, there was significantly increased uptake of ⁶⁸Ga-A20FMDV2 focally in the irradiated region within the right lung, compared to sham-treated control animals and the unirradiated left lung. <h3>Conclusion</h3> α_vβ₆ integrin-targeted PET imaging using ⁶⁸Ga-A20FMDV2 can serve as a powerful tool to study the evolution of radiation-induced pulmonary fibrosis <i>in vivo</i>, with significant potential for clinical translation.

Cardiac Complications in Marfan Syndrome: A Review

Marfan syndrome (MFS) is a rare inherited disorder of the connective tissue with an autosomal dominant mode of inheritance which happens as a result of a mutation in the fibrillin-1 (FBN1) gene located on chromosome 15q21.1. This mutation results in the defective formation of microfibrils and increased levels of active transforming growth factor beta (TGFbeta), leading to defective connective tissue synthesis. These changes affect various parts of the body but most notably affected are the heart, eyes, and the musculoskeletal system. The standard presenting features of a person suffering from MFS are tall stature with a large arm span, kyphosis, congenital dislocation of the lens (ectopia lentis) and cardiovascular manifestations. The 2010 modified Ghent criteria are used to diagnose MFS on the basis of parameters such as cardiovascular, eye, and musculoskeletal disorders. The cardiovascular manifestations in a patient with MFS are the leading causes of mortality. The most common and dreaded complication is an aortic aneurysm and subsequent dissection. Cardiomyopathy and arrhythmia are also potential killers in such patients. This article aims to look at the various cardiac complications mentioned above and gain an understanding of their pathogenesis, incidence, and outcome. It also includes a brief overview of the rare complication post-Bentall graft infection, and its cause, diagnosis, and management. Various articles by several different authors from around the world were searched for information regarding the pathogenesis, incidence, and outcomes of these patients and are referenced below.

Liver transcriptomics reveals microRNA features of the host response in a mouse model of dengue virus infection

BackgroundOrgan dysfunction, especially liver injury, caused by dengue virus (DENV) infection has been associated with fatal cases in dengue patients around the world. However, the pathophysiological mechanisms of liver involvement in dengue remain unclear. There is accumulating evidence that miRNAs are playing an important role in regulating viral pathogenesis, and it can help in diagnostic and anti-viral therapies development. MethodsWe collected liver tissues of DENV-infected for small RNA sequencing to identify significantly different express miRNAs during dengue virus infection, and the identified target genes of these miRNAs were annotated by biological function and pathway enrichment. Results31 significantly altered miRNAs were identified, including 16 up-regulated and 15 down-regulated miRNAs. By performing a series of miRNA prediction and signaling pathway enrichment analyses, the down-regulated miRNAs of mmu-miR-484, mmu-miR-1247-5p and mmu-miR-6538 were identified to be the crucial miRNAs. Further analysis revealed that the inflammation and immune responses involving Hippo, PI3K-Akt, MAPK, Wnt, mTOR, TGF-beta, Tight junction, and Platelet activation were modulated collectively by these three key miRNAs during DENV infection. These pathways are considered to be closely associated with the pathogenic mechanism and treatment strategy of dengue patients. ConclusionThe miRNAs identified by sequencing, especially miR-484 may be the potential therapeutic targets for liver involvement in dengue patients which involves the regulation of vascular permeability and expression of inflammatory cytokines.

Functional characterization of nutraceuticals using spectral clustering: Centrality of caveolae-mediated endocytosis for management of nitric oxide and vitamin D deficiencies and atherosclerosis.

It is well recognized that redox imbalance, nitric oxide (NO), and vitamin D deficiencies increase risk of cardiovascular, metabolic, and infectious diseases. However, clinical studies assessing efficacy of NO and vitamin D supplementation have failed to produce unambiguous efficacy outcomes suggesting that the understanding of the pharmacologies involved is incomplete. This raises the need for using systems pharmacology tools to better understand cause-effect relationships at biological systems levels. We describe the use of spectral clustering methodology to analyze protein network interactions affected by a complex nutraceutical, Cardio Miracle (CM), that contains arginine, citrulline, vitamin D, and antioxidants. This examination revealed that interactions between protein networks affected by these substances modulate functions of a network of protein complexes regulating caveolae-mediated endocytosis (CME), TGF beta activity, vitamin D efficacy and host defense systems. Identification of this regulatory scheme and the working of embedded reciprocal feedback loops has significant implications for treatment of vitamin D deficiencies, atherosclerosis, metabolic and infectious diseases such as COVID-19.

CWHM-12, an Antagonist of Integrin-Mediated Transforming Growth Factor-Beta Activation Confers Protection During Early Mycobacterium tuberculosis Infection in Mice.

Tuberculosis (TB) caused by the pathogenic bacterium Mycobacterium tuberculosis (Mtb) is one of the most lethal infectious diseases in the world. Presently, Bacillus Calmette-Guerin, the vaccine approved for use against TB, does not offer complete protection against the disease, which necessitates the development of new therapeutics to treat this infection. Overexpression of transforming growth factor beta (TGF-β) is associated with pulmonary profibrotic changes. The inactive TGF-β secreted is activated through its cleavage and release by αv integrins. Integrin-mediated regulation of TGF-β is considered as a master switch in the profibrotic process and a potential therapeutic target. Thus, in this study, we sought to determine if treatment with a broad range antagonist of integrins, CWHM-12, has the potency to inhibit pulmonary fibrosis and enhance Mtb control in a highly susceptible mouse model of Mtb infection, namely the C3Heb/FeJ (FeJ). CWHM-12 treatment at the early stages of Mtb infection was efficacious in reducing disease severity and inflammation associated with decreased iNOS, MIP-2, and IL-10 production without degradation of collagen. This suggests a potential for CWHM-12 targeting of TGF-β to be explored as an adjunct therapeutic for early Mtb infection.

Autophagy-Related Activation of Hepatic Stellate Cells Reduces Cellular miR-29a by Promoting Its Vesicular Secretion.

Background & AimsLiver fibrosis arises from long-term chronic liver injury, accompanied by an accelerated wound healing response with interstitial accumulation of extracellular matrix (ECM). Activated hepatic stellate cells (HSC) are the main source for ECM production. MicroRNA29a (miR-29a) is a crucial antifibrotic miRNA that is repressed during fibrosis, resulting in up-regulation of collagen synthesis.MethodsIntracellular and extracellular miRNA levels of primary and immortalized myofibroblastic HSC in response to profibrogenic stimulation by transforming growth factor β (TGFβ) or platelet-derived growth factor-BB (PDGF-BB) or upon inhibition of vesicular transport and autophagy processes were determined by quantitative polymerase chain reaction. Autophagy flux was studied by electron microscopy, flow cytometry, immunoblotting, and immunocytochemistry. Hepatic and serum miR-29a levels were quantified by using both liver tissue and serum samples from a cohort of chronic hepatitis C virus patients and a murine CCl4 induced liver fibrosis model.ResultsIn our study, we show that TGFβ and PDGF-BB resulted in decrease of intracellular miR-29a and a pronounced increase of vesicular miR-29a release into the supernatant. Strikingly, miR-29a vesicular release was accompanied by enhanced autophagic activity and up-regulation of the autophagy marker protein LC3. Moreover, autophagy inhibition strongly prevented miR-29a secretion and repressed its targets’ expression such as Col1A1. Consistently, hepatic miR-29a loss and increased LC3 expression in myofibroblastic HSC were associated with increased serum miR-29a levels in CCl4-treated murine liver fibrosis and specimens of hepatitis C virus patients with chronic liver disease.ConclusionsWe provide evidence that activation-associated autophagy in HSC induces release of miR-29a, whereas inhibition of autophagy represses fibrogenic gene expression in part through attenuated miR-29a secretion.