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The glycocalyx on tumor cells has been recently identified as an important driver for cancer progression, possibly providing critical opportunities for treatment. Metastasis, in particular, is often the limiting step in the survival to cancer, yet our understanding of how tumor cells escape the vascular system to initiate metastatic sites remains limited. Using an in vitro model of the human microvasculature, we assess here the importance of the tumor and vascular glycocalyces during tumor cell extravasation. Through selective manipulation of individual components of the glycocalyx, we reveal a mechanism whereby tumor cells prepare an adhesive vascular niche by depositing components of the glycocalyx along the endothelium. Accumulated hyaluronic acid shed by tumor cells subsequently mediates adhesion to the endothelium via the glycoprotein CD44. Trans-endothelial migration and invasion into the stroma occurs through binding of the isoform CD44v to components of the sub-endothelial extra-cellular matrix. Targeting of the hyaluronic acid-CD44 glycocalyx complex results in significant reduction in the extravasation of tumor cells. These studies provide evidence of tumor cells repurposing the glycocalyx to promote adhesive interactions leading to cancer progression. Such glycocalyx-mediated mechanisms may be therapeutically targeted to hinder metastasis and improve patient survival.Offeddu et al. employ a microfluidic vascular model to study the effects of selective removal of glycocalyx components from cancer cells. They find that cancer-associated hyaluronic acid and CD44 mediate firm adhesion to endothelial cells and extravasation during metastasis.

The genetically encoded GEVAL sensors allow ratiometric imaging of the spatiotemporal dynamics of cellular GTP levels in living cells. GTP is a major regulator of multiple cellular processes, but tools for quantitative evaluation of GTP levels in live cells have not been available. We report the development and characterization of genetically encoded GTP sensors, which we constructed by inserting a circularly permuted yellow fluorescent protein (cpYFP) into a region of the bacterial G protein FeoB that undergoes a GTP-driven conformational change. GTP binding to these sensors results in a ratiometric change in their fluorescence, thereby providing an internally normalized response to changes in GTP levels while minimally perturbing those levels. Mutations introduced into FeoB to alter its affinity for GTP created a series of sensors with a wide dynamic range. Critically, in mammalian cells the sensors showed consistent changes in ratiometric signal upon depletion or restoration of GTP pools. We show that these GTP evaluators (GEVALs) are suitable for detection of spatiotemporal changes in GTP levels in living cells and for high-throughput screening of molecules that modulate GTP levels.

BackgroundVascular endothelial growth factor-C (VEGF-C) expression and subsequent lymphangiogenesis in the tumor microenvironment are associated with metastasis and poor prognosis in melanoma and other solid tumors. We previously demonstrated that while elevated VEGF-C promotes a highly immunosuppressive tumor microenvironment (TME), it paradoxically renders immunotherapy more effective in mouse models and correlates with improved survival after checkpoint blockade therapy in patients, a phenomenon we termed lymphangiogenic potentiation.1 This potentiation correlated with VEGFC-driven differences in the TME prior to immunotherapy, including increased recruitment of naïve T cells and cross-presenting CD103+ DCs in VEGFC-overexpressing tumors, setting up the TME for local T cell activation .Other than altering the TME, tumor lymphangiogenesis may alter the systemic immune response because of increased drainage of tumor-secreted factors, which may include both immunogenic and suppressive factors, to the tumor-draining lymph node (tdLN). Here, we asked whether and how these changes may contribute to the overall immune response as well as how the anti-tumor immune response can alter the TME in melanoma.MethodsWe used a B16F10 tumor model transduced to either overexpress VEGFC (B16-VEGFC) or a control vector (B16-Control) together with two different models of immunotherapy: (i) adoptive T cell transfer and (ii) peptide vaccination using transferred naïve antigen-specific pmel CD8+ T cells for in vivo tracking after activation by gp100 peptide and CpG adjuvant.ResultsWe found that immunotherapy resulted in an increase targeted antigen-specific T cells in the B16-VEGFC vs. B16-Control tumors, including more proliferating effector cells as well as more central memory and T progenitor exhausted cells. We also found more bystander activation and proliferation of non-targeted, endogenous CD8 T cells. This was accompanied by an increase in CXCL9 in the TME of B16-VEGFC, which recruited more activated T cells from circulation. In the B16-VEGFC tdLN, we observed increased in CD103+ DC trafficking along with higher fractions of central memory T cells in both the CD4 and CD8 compartments.ConclusionsTogether, these results highlight the multitude of ways that tumor lymphangiogenesis can prime anti-tumor immunity and alter not only the tumor microenvironment, but also the systemic immune response by shaping the immune microenvironment of the tumor draining lymph node. VEGFC tumors promote more prolonged immunity after immunotherapy by supporting T cell memory development and enhancing ongoing immune activation and recruitment through increased DC trafficking to the lymph node and increased recruitment of newly activated and circulating T cells.ReferenceFankhauser M. Tumor lymphangiogenesis promotes T cell infiltration and potentiates immunotherapy in melanoma. Sci. Transl. Med. 2017;9:eaa14712.

Background The standard of care (SOC) treatment for drug-susceptible pulmonary tuberculosis (DS-TB) consists of isoniazid, rifampicin, pyrazinamide, and ethambutol (HRZE). New treatment regimen options for DS-TB are needed as HRZE is long in duration (6 months), associated with frequent adverse events, unforgiving of adherence lapses, and complicated by rifamycin-based drug-drug interactions. The recent resurgence of TB drug development, particularly in the context of drug-resistant TB, offers promise for additional regimens for persons with DS-TB, provided they are sufficiently effective and well-tolerated. We spotlight wave 1 of the RAD-TB platform trial (ACTG A5409, NCT06192160) that will investigate new chemical entities for the treatment of DS-TB. Methods In wave 1 of the RAD-TB platform, adult participants initiating treatment for DS-TB will be randomized to SOC (HRZE, Arm 1) or one of five experimental arms for the 8-week intensive phase. The experimental treatment arms will consist of a bedaquiline and pretomanid backbone (BPa) in combination with one of three oxazolidinones. Arm 2 will study linezolid (BPaL) at a dose of 600 mg daily, Arms 3A and 3B will study TBI-223 at 1200 mg and 2400 mg daily, respectively, and Arms 4A and 4B will study sutezolid at 800 mg and 1600 mg daily, respectively. The primary efficacy objective is to compare sputum culture time to positivity (TTP) slope over the first 6 weeks of treatment for each experimental treatment arm to SOC. The primary safety objective is to compare new Grade 3 or higher adverse events over the first 8 weeks of treatment for each experimental treatment arm to SOC. After the intensive phase, all participants will receive the standard isoniazid and rifampicin (HR) continuation phase for 18 weeks. Participants will be followed for 52 weeks after TB treatment initiation to assess long-term outcomes. Discussion Wave 1 of the RAD-TB platform aims to identify the optimal oxazolidinone(s), with regard to both efficacy and safety, to combine with the BPa backbone for the treatment of DS-TB. Subsequent waves of this platform trial may add a fourth drug to the regimen, study new diarylquinolines to substitute for bedaquiline, or study novel agents from other TB drug classes. Trials registration ClinicalTrials.gov NCT06192160 . Registered on January 5, 2024.

BackgroundAdoptive cell therapy with tumor-infiltrating lymphocytes (TILs) has demonstrated tremendous promise in clinical trials for patients with solid or metastatic tumors.1 However, current TIL therapy requires systemic administration of IL-2 to promote TIL survival, and IL-2-associated toxicities greatly limit patient eligibility and reduce the long-term clinical benefit of TIL therapy.2 3 Unlike IL-2, which promotes T cell exhaustion, IL-15 maintains antigen-independent TIL persistence through homeostatic proliferation and supports CD8+ T cell anti-tumor activity without stimulating regulatory T cells. We designed genetically engineered TILs to express a regulated form of membrane-bound IL-15 (mbIL15) for tunable long-term persistence, leading to enhanced efficacy and safety for the treatment of patients with solid tumors.MethodsObsidian’s cytoDRiVE™ platform includes small human protein sequences called drug responsive domains (DRD)s that enable regulated expression of a fused target protein under control of FDA-approved, bioavailable small molecule ligands. cytoTIL15 contains TILs engineered with mbIL15 under the control of a carbonic-anhydrase-2 DRD, controlled by the ligand acetazolamide (ACZ). After isolation from tumors, TILs were transduced and expanded in vitro through a proprietary TIL expansion process. cytoTIL15 were immunophenotyped and assessed for in vitro antigen-independent survival and co-cultured with tumor cells to assess polyfunctionality and cytotoxicity. In vivo TIL persistence and anti-tumor efficacy was evaluated through adoptive transfer of TILs into immunodeficient NSG mice, either naïve or implanted with subcutaneous patient-derived-xenograft (PDX) tumors.Results cytoTIL15 and conventional IL2-dependent TILs isolated from melanoma tumor samples expanded to clinically relevant numbers over 14 days. Throughout expansion, cytoTIL15 were enriched for CD8+ T cells and acquired enhanced memory-like characteristics, while maintaining diverse TCRVβ sub-family representation. cytoTIL15 demonstrated enhanced potency over conventional TILs, as measured by increased polyfunctionality and cytotoxicity against tumor and PDX lines in vitro (figure 1A). In a 10-day antigen-independent in vitro assay, cytoTIL15 persisted at greater frequencies than conventional TILs in the absence of IL-2 (figure 1B; *p<0.05). cytoTIL15 adoptively transferred into naïve NSG mice demonstrated ACZ-dependent long-term persistence without antigen or exogenous IL-2, whereas conventional TILs were undetectable >30 days following adoptive cell transfer (figure 1C). Importantly, cytoTIL15 achieved significant tumor control in a human PDX model (figure 1D), which correlated with increased TIL accumulation in secondary lymphoid organs.Abstract 166 Figure 1cytoTIL15 demonstrate superior persistence. cytoTIL15 is an engineered TIL product expressing regulatable mbIL15. (A) cytoTIL15 demonstrate enhanced in vitro cytotoxicity after co-culture with melanoma tumor lines (representative data from 3 TIL donors). (B) cytoTIL15 have improved persistence in antigen- and IL2- independent culture conditions in vitro compared to conventional TILs cultured in the absence of IL-2 as well as (C) in vivo compared to conventional TILs supplemented with IL-2, when engrafted into NSG mice (in vitro: representative data from 1 TIL donor, performed in >3 replicate donors, in vivo: n=5/group, representative of 1 TIL donor, performed in >3 replicate donors). (D) cytoTIL15 (with 200mg/kg ACZ PO QD) demonstrate enhanced anti-tumor efficacy in a xenograft melanoma model as compared to conventional TILs (with 50000 IU IL-2 q8h BID, IP for 5 days) (n=8/group, representative of 1 TIL donor, performed in >2 replicate donors; ACT = adoptive cell transfer).ConclusionsTaken together, the superior persistence and potency of cytoTIL15 in the complete absence of IL-2 highlights the clinical potential of cytoTIL15 as a novel TIL product with enhanced safety and efficacy for patients with melanomas, and other solid tumors.AcknowledgementsThe authors wish to acknowledge the Cooperative Human Tissue Network for the their supply of human tumor tissue, and the MD Anderson Cancer Center for technical support; schematic created with BioRender.com.ReferencesChandran SS, Somerville RPT, Yang JC, Sherry RM, Klebanoff CA, Goff SL, Wunderlich JR, Danforth DN, Zlott D, Paria BC, Sabesan AC, Srivastava AK, Xi L, Pham TH, Raffeld M, White DE, Toomey MA, Rosenberg SA, Kammula US. Treatment of metastatic uveal melanoma with adoptive transfer of tumour-infiltrating lymphocytes: a single-centre, two-stage, single-arm, phase 2 study. Lancet Oncol 2017 Jun;18(6):792–802. doi: 10.1016/S1470-2045(17)30251-6. Epub 2017 Apr 7. PMID: 28395880; PMCID: PMC5490083.Yang JC. Toxicities associated with adoptive T-cell transfer for Cancer. Cancer J 2015;21:506–9.Schwartz RN, Stover L, Dutcher JP. Managing toxicities of high-dose interleukin-2. Oncology (Williston Park) 2002 Nov;16(11 Suppl 13):11–20. PMID: 12469935.

Interleukin-10 (IL-10) is a potent immunoregulatory cytokine that suppresses pro-inflammatory cytokine production, reduces antigen presentation by myeloid cells, promotes M2 macrophage polarization, and inhibits T cell activation. Despite these well-established immunoregulatory functions, efforts to harness recombinant IL-10 therapeutically have been limited by its short plasma half-life and poor retention in the secondary lymphoid organs (SLOs), key sites of autoreactive T cell priming in autoimmune disease. Previously, we engineered a fusion of serum albumin and IL-10 (SA-IL-10) with extended half-life and enhanced exposure in the SLOs following intravenous administration. Here, we integrate human transcriptomic analyses and a murine model of neuroinflammation, experimental autoimmune encephalomyelitis (EAE), to investigate how sustained IL-10 exposure in the SLOs modulates immune responses under inflammatory conditions. Human single-cell RNA sequencing analyses revealed reduced IL-10 expression alongside increased IL-10 receptor expression across multiple immune cell populations in treatment-naïve patients with multiple sclerosis (MS), motivating the investigation of IL-10-based immunomodulatory strategies. Prophylactic SA-IL-10 administration prevented the development and progression of EAE with superior efficacy to wild type IL-10 and comparable protection to fingolimod, an FDA-approved MS therapy. Immunophenotyping of the SLOs revealed that SA-IL-10 suppressed pathogenic, antigen-specific RORγt Foxp3 T 17 T cells, CD86 M1-like macrophages, CD86 dendritic cells, and pro-inflammatory cytokine production, while expanding immunoregulatory CD206 M2-like macrophages and increasing the frequency of multiple checkpoint markers (CTLA-4, PD-1, TIGIT, ICOS) on GATA3 Foxp3 T 2 cells. Despite the absence of direct central nervous system targeting, SA-IL-10 treatment also reduced the infiltration of macrophages, dendritic cells, and CD4 T cells into the spinal cord. Repeated SA-IL-10 administration was well tolerated, as treated EAE mice gained significantly more body weight over the course of treatment compared to PBS- and WT IL-10-treated controls, and exhibited plasma biochemistry parameters comparable to control animals at study endpoint. Together, these findings demonstrate that increasing IL-10 exposure in the SLOs suppresses neuroinflammation by promoting immunoregulation. Subcutaneously administered serum albumin-fused interleukin-10 prevents experimental autoimmune encephalomyelitis by suppressing pathogenic T 17 cells and pro-inflammatory myeloid cells in the secondary lymphoid organs and spinal cord, while expanding immunoregulatory cells in the secondary lymphoid organs.

Melanoma progression is associated with increased invasion and, often, decreased levels of microphthalmia-associated transcription factor (MITF). Accordingly, downregulation of MITF induces invasion in melanoma cells; however, little is known about the underlying mechanisms. Here, we report for the first time that depletion of MITF results in elevation of intracellular GTP levels and increased amounts of active (GTP-bound) RAC1, RHO-A and RHO-C. Concomitantly, MITF-depleted cells display larger number of invadopodia and increased invasion. We further demonstrate that the gene for guanosine monophosphate reductase ( GMPR ) is a direct MITF target, and that the partial repression of GMPR accounts mostly for the above phenotypes in MITF-depleted cells. Reciprocally, transactivation of GMPR is required for MITF-dependent suppression of melanoma cell invasion, tumorigenicity and lung colonization. Moreover, loss of GMPR accompanies downregulation of MITF in vemurafenib-resistant BRAF V600E -melanoma cells and underlies the increased invasion in these cells. Our data uncover novel mechanisms linking MITF-dependent inhibition of invasion to suppression of guanylate metabolism.

IntroductionLimited data exist on US referral/management patterns for moderate-to-severe thyroid eye disease (TED), a disabling condition. MethodsUS ophthalmologists and endocrinologists experienced in treating TED provided medical record data of moderate-to-severe TED patients and information on referral/treatment practices. Data on signs/symptoms, medical/surgical treatments, treatment response, and referral history were collected. Moderate and severe cases were stratified to interrogate treatment/practice differences. ResultsA total of 181 physicians provided data on 714 patients (49.4 ± 13.6 years old, 65% women, 14% severe disease). Reporting physicians diagnosed 55% of patients themselves and solely managed 37% of cases, with similar referral/comanagement patterns between moderate and severe cases. Topical therapies included lubricating (79%) and glucocorticoid (39%) eye drops. Systemic therapies included oral glucocorticoids (36%), IV glucocorticoids (15%), and rituximab and/or tocilizumab (12%). Few patients underwent orbital radiation (4%) or surgical intervention (4%). IV glucocorticoids (33% vs. 12%), biologics (26% vs. 10%), orbital radiation (11% vs. 3%), and ocular surgery (12% vs. 3%) were used more often in severe versus moderate cases (all P < 0.001). However, severe disease was less responsive to therapy (very responsive to therapy: 28% vs. 49%, P < 0.001). ConclusionsParticipating physicians were primarily responsible for just over one-half of TED diagnoses, but solely treated <40% of patients. Severe TED was treated more often with surgery and systemic immunologic therapies than moderate disease, but was less likely to respond to treatment. These results reinforce that moderate-to-severe TED is difficult to treat with an unmet medical need in the United States.

Widowed fathers with dependent children face a complex situation as they manage their expanded parenting responsibilities as the surviving parent while grieving for their spouse. Since there are specific parenting roles that mothers and fathers are expected to fulfill that are largely shaped and reinforced by gender role socialization and norms, it is particularly expected that challenges may arise when widowed fathers need to fulfill aspects of the caregiver-nurturer parental role that are not traditionally assigned to men. The literature reveals very limited research focusing on the unique experiences and needs of widowed fathers with dependent children. The importance of gaining a better understanding of how widowed fathers adjust to single parent role has been emphasized by research findings indicating that widowed parents' coping and parenting approaches and overall adjustment can have a significant impact on their children's well-being. This study employed qualitative conventional content analysis that allowed the researcher to engage in an in-depth examination of the experiences of eight widowed fathers and how they made adjustments to their parenting role after the death of their spouse. The findings of this study indicate that widowed fathers have to undergo a significant process of adjustment as they assume the single father role. It appears that widowed fathers have to continually reshape their role and responsibilities as a single parent, as a male, and as a person, as they balance their parenting and provider roles after the death of their spouse. The gender specific parenting roles and gender role norms that are still prevalent in our society also appear to inform aspects of the widowed fathers' experiences of taking on the primary parent role as well as the nature of the challenges they faced in doing so. Additional themes from the data included: relationship between prior paternal involvement and difficulty of adjustment, fathers' sense of children's need for continuity and routines, shifts in parenting style during adjustment, and unexpected changes in fathers' sense of self. Findings are discussed with regard to clinical implications for working with widowed fathers with dependent children, and recommendations for future research with this population are provided.

MOVIEPASS CLASS ACTION LAWSUIT MoviePass, an entertainment company that advertised a new way to attend movies through a subscription service initially allowing members to attend a number of movies per month for a flat monthly fee, is now faced with a shareholder class action lawsuit filed in August in New York federal district court against its parent company, Helios and Matheson Analytics, Inc. After Helios acquired a majority stake in MoviePass last year, a new business model was introduced which allowed subscribers to see unlimited movies for only $9.95 per month. [...]the Court rejected a misuse defense by Redbox due to the changes Disney made to the various terms of use on the download websites. [...]parties involved in any speculation or hard evidence will not meet the requirements of collusion. Article 17 limits the collusion investigation to conduct of the NFL and its teams. [...]Kaepernick's deal with Nike would likely be outside the scope of collusion.