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Background: Immune checkpoint inhibitors (ICIs) have moved to the frontline in recent years to manage upper gastrointestinal (UGI) tumors, such as esophageal and gastric cancers. This retrospective review sheds light on real-world data on ICI-treated UGI tumors to identify risk factors (clinical and pathological) impacting the outcome other than traditional biomarkers (programmed cell death ligand 1 (PD-L1) or microsatellite instability status)., Methods: Patients with UGI tumors who received at least one dose of ICI for stage IV or recurrent disease between January 1, 2015, and July 31, 2021, at The Ohio State University were included in the study. The patients' baseline characteristics, labs, and blood counts (even at disease progression) were extracted with survival outcomes (progression-free survival (PFS) and overall survival (OS)). Descriptive statistics, log-rank test and Cox proportional hazard model for survival outcomes, Fisher exact test for categorical variables, were conducted using JMP Pro 16 (SAS Institute Inc., Cary, NC)., Results: We had 64 patients (84% males) included in the study, with the racial distribution as follows: 88% Caucasian, 5% African American, 1% Asian, and 6% from other racial groups. Men and the use of ICI in third lines or more had a positive impact on PFS and OS. For OS, 1) history of surgery positively impacted the outcome, while bone metastases worsened it; 2) baseline red blood cell count (RBC), hemoglobin, and thyroid-stimulating hormone (TSH) negatively impacted the OS. For PFS, 1) PD-L1 positivity, baseline lymphocyte count, and aspartate transferase levels had a positive impact; 2) human epidermal growth factor receptor 2 (HER2) positivity, baseline RBC, TSH, alkaline phosphatase, and alanine transferase (AST) levels had a negative impact. A slight increase in white blood cell (WBC) count (by 1.54, P = 0.02) and a drop in lymphocyte count (by 0.1907, P = 0.003) was significantly associated with disease progression., Conclusions: Baseline risk factors and monitoring blood counts can help predict outcomes in ICI-treated UGI tumors. We need larger studies to confirm this., Competing Interests: The authors declare no conflict of interest., (Copyright 2024, Manne et al.)

Neoadjuvant therapy (NAT) for early-stage pancreatic ductal adenocarcinoma (PDA) has recently gained prominence. We investigated the clinical significance of mucin 5 AC (MUC5AC), which exists in two major glycoforms, a less-glycosylated immature isoform (IM) and a heavily glycosylated mature isoform (MM), as a biomarker in resected PDA. Immunohistochemistry was performed on 100 resected PDAs to evaluate the expression of the IM and MM of MUC5AC using their respective monoclonal antibodies, CLH2 (NBP2-44455) and 45M1 (ab3649). MUC5AC localization (cytoplasmic, apical, and extra-cellular (EC)) was determined, and the H-scores were calculated. Univariate and multivariate (MVA) Cox regression models were used to estimate progression-free survival (PFS) and overall survival (OS). Of 100 resected PDA patients, 43 received NAT, and 57 were treatment-naïve with upfront surgery (UpS). In the study population ( n = 100), IM expression (H-scores for objective response vs. no response vs. UpS = 104 vs. 152 vs. 163, p = 0.01) and MM-MUC5AC detection rates (56% vs. 63% vs. 82%, p = 0.02) were significantly different. In the NAT group, MM-MUC5AC-negative patients had significantly better PFS according to the MVA (Hazard Ratio: 0.2, 95% CI: 0.059-0.766, p = 0.01). Similar results were noted in a FOLFIRINOX sub-group ( n = 36). We established an association of MUC5AC expression with treatment response and outcomes.

Background: Hepatocellular carcinoma (HCC) disproportionately impacts racial and ethnic minorities and patients with lower socioeconomic status. These social determinants of health (SDH) lead to disparities in access to care and outcomes. We aim to understand the relationship between SDH and survival and locoregional treatment options in HCC., Methods: Using the National Cancer Database, we evaluated survival and access locoregional treatments including non-transplant surgery, liver transplant (LT), and liver-directed radiation therapy (LDRT) in patients with HCC diagnosed between 2004 and 2017. Variables including clinical stage, age, sex, race, income, rurality, year of diagnosis, facility type (FT), Charlson-Deyo score (CD), and insurance were evaluated. Cox proportional hazards multivariable regression and dominance analyses were used for analyses., Results: In total, 140 340 patients were included. Worse survival was seen with advanced stage, older age, Black race, rurality, public insurance, treatment at a nonacademic center, and lower income. The top predictors for survival included stage, age, and income. Completion of non-transplant surgery was best predicted by stage, FT, and insurance type, whereas LT was predicted by age, year of diagnosis, and CD score. LDRT utilization was most associated with year of diagnosis, FT, and CD score., Conclusion: For patients with HCC, survival was predicted primarily by stage, age, and income. The primary sociodemographic factors associated with access to surgical treatments, in addition to FT, were insurance and income, highlighting the financial burdens of health care. Work is needed to address disparities in access to care, including improved insurance access, addressing financial inequities and financial toxicities of treatments, and equalizing care opportunities in community centers., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Tumor evolution from a single cell into a malignant, heterogeneous tissue remains poorly understood. Here, we profile single-cell transcriptomes of genetically engineered mouse lung tumors at seven stages, from pre-neoplastic hyperplasia to adenocarcinoma. The diversity of transcriptional states increases over time and is reproducible across tumors and mice. Cancer cells progressively adopt alternate lineage identities, computationally predicted to be mediated through a common transitional, high-plasticity cell state (HPCS). Accordingly, HPCS cells prospectively isolated from mouse tumors and human patient-derived xenografts display high capacity for differentiation and proliferation. The HPCS program is associated with poor survival across human cancers and demonstrates chemoresistance in mice. Our study reveals a central principle underpinning intra-tumoral heterogeneity and motivates therapeutic targeting of the HPCS., Competing Interests: Declaration of Interests T.J. is a member of the Board of Directors of Amgen and Thermo Fisher Scientific, and a co-Founder of Dragonfly Therapeutics and T2 Biosystems. T.J. serves on the Scientific Advisory Board of Dragonfly Therapeutics, SQZ Biotech, and Skyhawk Therapeutics. T.J. also received funding from Calico and currently receives funding from Johnson & Johnson, but this funding did not support the research described in this manuscript. A.R. is a co-founder and equity holder in Celsius Therapeutics and a SAB member for Thermo Fisher, Asimov, Neogene Therapeutics, and Syros Pharmaceuticals, and an equity holder of Immunitas Therapeutics. C.M.R. serves on the SAB of Bridge Medicines and Harpoon Therapeutics, and has consulted regarding oncology drug development with AbbVie, Amgen, Ascentage, Bicycle, Celgene, Daiichi Sankyo, Genentech, Ipsen, Loxo, Pharmamar, and Vavotek. None of the affiliations listed above represent a conflict of interest with the design or execution of this study or interpretation of data presented in this manuscript. Other authors have nothing to disclose., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Glioblastoma is the most frequently occurring and invariably fatal primary brain tumor in adults. The vast majority of glioblastomas is characterized by chromosomal copy number alterations, including gain of whole chromosome 7 and loss of whole chromosome 10. Gain of whole chromosome 7 is an early event in gliomagenesis that occurs in proneural-like precursor cells, which give rise to all isocitrate dehydrogenase (IDH) wild-type glioblastoma transcriptional subtypes. Platelet-derived growth factor A ( PDGFA ) is one gene on chromosome 7 known to drive gliomagenesis, but, given its location near the end of 7p, there are likely several other genes located along chromosome 7 that select for its increased whole-chromosome copy number within glioblastoma cells. To identify other potential genes that could select for gain of whole chromosome 7, we developed an unbiased bioinformatics approach that identified homeobox A5 ( HOXA5 ) as a gene whose expression correlated with gain of chromosome 7 and a more aggressive phenotype of the resulting glioma. High expression of HOXA5 in glioblastoma was associated with a proneural gene expression pattern and decreased overall survival in both human proneural and PDGF-driven mouse glioblastoma. Furthermore, HOXA5 overexpression promoted cellular proliferation and potentiated radioresistance. We also found enrichment of HOXA5 expression in recurrent human and mouse glioblastoma at first recurrence after radiotherapy. Overall, this study implicates HOXA5 as a chromosome 7-associated gene-level locus that promotes selection for gain of whole chromosome 7 and an aggressive phenotype in glioblastoma., (© 2018 Cimino et al.; Published by Cold Spring Harbor Laboratory Press.)

Glioblastomas are the most aggressive primary brain tumor. Despite treatment with surgery, radiation and chemotherapy, these tumors remain uncurable and few significant increases in survival have been observed over the last half-century. We recently employed a combined theoretical and experimental approach to predict the effectiveness of radiation administration schedules, identifying two schedules that led to superior survival in a mouse model of the disease (Leder et al., Cell 156(3):603-616, 2014). Here we extended this approach to consider fractionated schedules to best minimize toxicity arising in early- and late-responding tissues. To this end, we decomposed the problem into two separate solvable optimization tasks: (i) optimization of the amount of radiation per dose, and (ii) optimization of the amount of time that passes between radiation doses. To ensure clinical applicability, we then considered the impact of clinical operating hours by incorporating time constraints consistent with operational schedules of the radiology clinic. We found that there was no significant loss incurred by restricting dosage to an 8:00 a.m. to 5:00 p.m. window. Our flexible approach is also applicable to other tumor types treated with radiotherapy.

The current difficulty in visualizing the true extent of malignant brain tumors during surgical resection represents one of the major reasons for the poor prognosis of brain tumor patients. Here, we evaluated the ability of a hand-held Raman scanner, guided by surface-enhanced Raman scattering (SERS) nanoparticles, to identify the microscopic tumor extent in a genetically engineered RCAS/tv-a glioblastoma mouse model. In a simulated intraoperative scenario, we tested both a static Raman imaging device and a mobile, hand-held Raman scanner. We show that SERS image-guided resection is more accurate than resection using white light visualization alone. Both methods complemented each other, and correlation with histology showed that SERS nanoparticles accurately outlined the extent of the tumors. Importantly, the hand-held Raman probe not only allowed near real-time scanning, but also detected additional microscopic foci of cancer in the resection bed that were not seen on static SERS images and would otherwise have been missed. This technology has a strong potential for clinical translation because it uses inert gold-silica SERS nanoparticles and a hand-held Raman scanner that can guide brain tumor resection in the operating room.

Glioblastomas (GBMs) are the most common and malignant primary brain tumors and are aggressively treated with surgery, chemotherapy, and radiotherapy. Despite this treatment, recurrence is inevitable and survival has improved minimally over the last 50 years. Recent studies have suggested that GBMs exhibit both heterogeneity and instability of differentiation states and varying sensitivities of these states to radiation. Here, we employed an iterative combined theoretical and experimental strategy that takes into account tumor cellular heterogeneity and dynamically acquired radioresistance to predict the effectiveness of different radiation schedules. Using this model, we identified two delivery schedules predicted to significantly improve efficacy by taking advantage of the dynamic instability of radioresistance. These schedules led to superior survival in mice. Our interdisciplinary approach may also be applicable to other human cancer types treated with radiotherapy and, hence, may lay the foundation for significantly increasing the effectiveness of a mainstay of oncologic therapy. PAPERCLIP:, (Copyright © 2014 Elsevier Inc. All rights reserved.)

Cancer cells subvert the natural balance between cellular life and death, achieving immortality through pathologic enforcement of survival pathways and blockade of cell death mechanisms. Pro-apoptotic BCL-2 family proteins are frequently disarmed in relapsed and refractory cancer through genetic deletion or interaction-based neutralization by overexpressed antiapoptotic proteins, resulting in resistance to chemotherapy and radiation treatments. New pharmacologic strategies are urgently needed to overcome these formidable apoptotic blockades. We harnessed the natural killing activity of BCL-2-interacting mediator of cell death (BIM), which contains one of the most potent BH3 death domains of the BCL-2 protein family, to restore BH3-dependent cell death in resistant hematologic cancers. A hydrocarbon-stapled peptide modeled after the BIM BH3 helix broadly targeted BCL-2 family proteins with high affinity, blocked inhibitory antiapoptotic interactions, directly triggered proapoptotic activity, and induced dose-responsive and BH3 sequence-specific cell death of hematologic cancer cells. The therapeutic potential of stapled BIM BH3 was highlighted by the selective activation of cell death in the aberrant lymphoid infiltrates of mice reconstituted with BIM-deficient bone marrow and in a human AML xenograft model. Thus, we found that broad and multimodal targeting of the BCL-2 family pathway can overcome pathologic barriers to cell death.

The difficulty in delineating brain tumor margins is a major obstacle in the path toward better outcomes for patients with brain tumors. Current imaging methods are often limited by inadequate sensitivity, specificity and spatial resolution. Here we show that a unique triple-modality magnetic resonance imaging-photoacoustic imaging-Raman imaging nanoparticle (termed here MPR nanoparticle) can accurately help delineate the margins of brain tumors in living mice both preoperatively and intraoperatively. The MPRs were detected by all three modalities with at least a picomolar sensitivity both in vitro and in living mice. Intravenous injection of MPRs into glioblastoma-bearing mice led to MPR accumulation and retention by the tumors, with no MPR accumulation in the surrounding healthy tissue, allowing for a noninvasive tumor delineation using all three modalities through the intact skull. Raman imaging allowed for guidance of intraoperative tumor resection, and a histological correlation validated that Raman imaging was accurately delineating the brain tumor margins. This new triple-modality-nanoparticle approach has promise for enabling more accurate brain tumor imaging and resection.

Post-transcriptional regulation of gene expression contributes to the protein output of a cell, however, methods for measuring translational regulation in complex in vivo systems are lacking. Here, we describe a sensitive method for measuring translational regulation in defined cell populations from heterogeneous tissue in vivo. We adapted the translating ribosome affinity purification (TRAP) methodology to measure the relative occupancy of individual mRNA transcripts in translating ribosomes in the Olig2-positive tumor cell population in a genetically engineered mouse model (GEM) of glioma. Global measurement of paired ribosome-bound and total cellular mRNA populations from tumor cells in vivo identified a broad distribution of relative ribosome occupancies amongst mRNA species that was highly reproducible across biological samples. Comparison of the translation state of glioma cells to non-transformed oligodendrocyte progenitor cells in normal brain identified global alteration of translation in tumor, and specifically of genes involved in cell division and synthetic metabolism. Furthermore, investigation of alteration in steady state translational efficiencies upon loss of PTEN, one of the most frequently mutated and deleted tumor suppressors in glioma, identified differential translation of proteins involved in cellular respiration, canonically regulated by PI3K/Akt signaling, and cellular glycosylation profiles, deregulation of which is known to be associated with tumor progression. Application of the translation efficiency profiling method described here to other biological contexts and conditions would extend our knowledge of the scope and impact of this important mode of gene regulation in complex in vivo systems.

Background: The tumor microenvironment contains normal, non-neoplastic cells that may contribute to tumor growth and maintenance. Within PDGF-driven murine gliomas, tumor-associated astrocytes (TAAs) are a large component of the tumor microenvironment. The function of non-neoplastic astrocytes in the glioma microenvironment has not been fully elucidated; moreover, the differences between these astrocytes and normal astrocytes are unknown. We therefore sought to identify genes and pathways that are increased in TAAs relative to normal astrocytes and also to determine whether expression of these genes correlates with glioma behavior., Methodology/principal Findings: We compared the gene expression profiles of TAAs to normal astrocytes and found the Antigen Presentation Pathway to be significantly increased in TAAs. We then identified a gene signature for glioblastoma (GBM) TAAs and validated the expression of some of those genes within the tumor. We also show that TAAs are derived from the non-tumor, stromal environment, in contrast to the Olig2+ tumor cells that constitute the neoplastic elements in our model. Finally, we validate this GBM TAA signature in patients and show that a TAA-derived gene signature predicts survival specifically in the human proneural subtype of glioma., Conclusions/significance: Our data identifies unique gene expression patterns between populations of TAAs and suggests potential roles for stromal astrocytes within the glioma microenvironment. We show that certain stromal astrocytes in the tumor microenvironment express a GBM-specific gene signature and that the majority of these stromal astrocyte genes can predict survival in the human disease.

BAX is a pro-apoptotic protein of the BCL-2 family that is stationed in the cytosol until activated by a diversity of stress stimuli to induce cell death. Anti-apoptotic proteins such as BCL-2 counteract BAX-mediated cell death. Although an interaction site that confers survival functionality has been defined for anti-apoptotic proteins, an activation site has not been identified for BAX, rendering its explicit trigger mechanism unknown. We previously developed stabilized alpha-helix of BCL-2 domains (SAHBs) that directly initiate BAX-mediated mitochondrial apoptosis. Here we demonstrate by NMR analysis that BIM SAHB binds BAX at an interaction site that is distinct from the canonical binding groove characterized for anti-apoptotic proteins. The specificity of the human BIM-SAHB-BAX interaction is highlighted by point mutagenesis that disrupts functional activity, confirming that BAX activation is initiated at this novel structural location. Thus, we have now defined a BAX interaction site for direct activation, establishing a new target for therapeutic modulation of apoptosis.

The BCL-2 family of apoptotic proteins regulates the critical balance between cellular life and death and, thus, has become the focus of intensive basic science inquiry and a fundamental target for therapeutic development in oncology and other diseases. Classified based on the presence of conserved alpha-helical motifs and pro- and anti-apoptotic functionalities, BCL-2 proteins participate in a complex interaction network that determines cellular fate. The identification of BCL-2 homology domain 3 (BH3) as a critical death helix that engages and regulates BCL-2 family proteins has inspired the development of molecular tools to decode and drug the interaction network. Stabilized Alpha-Helices of BCL-2 domains (SAHBs) are structurally reinforced, protease-resistant, and cell-permeable compounds that retain the specificity of native BH3 death ligands and, therefore, serve as ideal reagents to dissect BCL-2 family interactions in vitro and in vivo. Here, we describe the in vitro and cell-based methods that exploit SAHB compounds to determine the functional consequences of BH3 interactions in regulating apoptosis.

Rational design of compounds to mimic the functional domains of BCL-2 family proteins requires chemical reproduction of the biologic complexity afforded by the relatively large and folded surfaces of BCL-2 homology (BH) domain peptide alpha-helices. Because the intermolecular handshakes of BCL-2 proteins are so critical to controlling cellular fate, we undertook the development of a toolbox of peptidic ligands that harness the natural potency and specificity of BH alpha-helices to interrogate and potentially medicate the deregulated apoptotic pathways of human disease. To overcome the classic deficiencies of peptide reagents, including loss of bioactive structure in solution, rapid proteolytic degradation in vivo, and cellular impermeability, we developed a new class of compounds based on hydrocarbon stapling of BH3 death domain peptides. Here we describe the chemical synthesis of Stabilized Alpha-Helices of BCL-2 domains or SAHBs, and the analytical methods used to characterize their secondary structure, proteolytic stability, and cellular penetrance.

The multidomain pro-apoptotic proteins BAX and BAK constitute an essential gateway to mitochondrial dysfunction and programmed cell death. Among the "BCL-2 homology (BH) 3-only" members of pro-apoptotic proteins, truncated BID (tBID) has been implicated in direct BAX activation, although an explicit molecular mechanism remains elusive. We find that BID BH3 peptide alone at submicromolar concentrations cannot activate BAX or complement BID BH3 mutant-tBID in mitochondrial and liposomal release assays. Because tBID contains structurally defined membrane association domains, we investigated whether membrane targeting of BID BH3 peptide would be sufficient to restore its pro-apoptotic activity. We developed a Ni(2+)-nitrilotriacetic acid liposomal assay system that efficiently conjugates histidine-tagged peptides to a simulated outer mitochondrial membrane surface. Strikingly, nanomolar concentrations of a synthetic BID BH3 peptide that is chemically tethered to the liposomal membrane activated BAX almost as efficiently as tBID itself. These results highlight the importance of membrane targeting of the BID BH3 domain in tBID-mediated BAX activation and support a model in which tBID engages BAX to trigger its pro-apoptotic activity.

BAX is a multidomain proapoptotic BCL-2 family protein that resides in the cytosol until activated by an incompletely understood trigger mechanism, which facilitates BAX translocation to mitochondria and downstream death events. Whether BAX is activated by direct contact with select BH3-only members of the BCL-2 family is highly debated. Here we detect and quantify a direct binding interaction between BAX and a hydrocarbon-stapled BID BH3 domain, which triggers the functional activation of BAX at nanomolar doses in vitro. Chemical reinforcement of BID BH3 alpha helicity was required to reveal the direct BID BH3-BAX association. We confirm the specificity of this BH3 interaction by characterizing a stapled BAD BH3 peptide that interacts with antiapoptotic BCL-X(L) but does not bind or activate BAX. We further demonstrate that membrane targeting of stapled BID BH3 optimizes its ability to activate BAX, supporting a model in which BID directly engages BAX to trigger mitochondrial apoptosis.