Palliative Care Needs of Patients with Musculoskeletal Malignancies.

PURPOSE OF REVIEW: This review aims to assess the literature regarding current treatment options for the palliative care of patients with advanced musculoskeletal malignancies whether primary or metastatic. RECENT FINDINGS: The inclusion of specialized palliative care physicians, in conjunction with surgeons, medical oncologists, radiation oncologists, interventional radiologists, and mental health professionals, results in better control of end-of-life symptoms in both children and adults with terminal musculoskeletal malignancies. The palliative care of patients with musculoskeletal malignancies requires a multi-disciplinary team and benefits from specialized palliative care physicians. The unique impacts of musculoskeletal malignancies on ambulation and independence creates additional mental and physical burdens on patients and care-takers alike. Palliative care should focus on preserving ambulatory function and patient independence, in addition to managing chronic pain and other end-of-life symptoms common to these malignancies.

Methods to Enable Spatial Transcriptomics of Bone Tissues.

Understanding the relationship between the cells and their location within each tissue is critical to uncover the biological processes associated with normal development and disease pathology. Spatial transcriptomics is a powerful method that enables the analysis of the whole transcriptome within tissue samples, thus providing information about the cellular gene expression and the histological context in which the cells reside. While this method has been extensively utilized for many soft tissues, its application for the analyses of hard tissues such as bone has been challenging. The major challenge resides in the inability to preserve good quality RNA and tissue morphology while processing the hard tissue samples for sectioning. Therefore, a method is described here to process freshly obtained bone tissue samples to effectively generate spatial transcriptomics data. The method allows for the decalcification of the samples, granting successful tissue sections with preserved morphological details while avoiding RNA degradation. In addition, detailed guidelines are provided for samples that were previously paraffin-embedded, without demineralization, such as samples collected from tissue banks. Using these guidelines, high-quality spatial transcriptomics data generated from tissue bank samples of primary tumor and lung metastasis of bone osteosarcoma are shown.

Utility of Indocyanine Green for Sentinel Lymph Node Biopsy in Pediatric Sarcoma and Melanoma.

BACKGROUND: Indocyanine green (ICG) is a fluorescent dye with increasing use for adult sentinel lymph node biopsy (SLNB). The utility of ICG in pediatric oncology remains understudied. We aim to describe our experience using ICG for SLNB in pediatrics versus standard blue dye. METHODS: A retrospective review of pediatric patients with melanoma or sarcoma who underwent SLNB with technetium plus ICG or blue dye from 2014 to 2023 at a large academic children's hospital was conducted. RESULTS: Twenty-four patients were included; 58.3% were male with median age 13 years (range 4-21 years). The majority had a melanocytic tumor (91.7%) and 8.3% had sarcoma. All patients received technetium with concomitant blue dye (62.5%) or ICG (37.5%). ICG more reliably identified radioactive SLNs, compared to blue dye (mean 100% vs 78.3 ± 8.3%, p = 0.03). There was no significant difference in median operative time (ICG 82 min [68-203] vs blue dye 93 min [78-105], p = 0.84). Seven patients had positive SLNs (29.2%), with recurrence in 2 patients (8.3%) and 1 death (4.2%). There were no adverse events. CONCLUSION: ICG-directed SLNB in children is a safe and effective alternative to blue dye. Use of ICG did not add to operative time, and more often identified sentinel nodes versus blue dye. TYPE OF STUDY: Original Research Article, Retrospective Comparative Study. LEVEL OF EVIDENCE: III.

RNA-sequencing predicts a role of androgen receptor and aldehyde dehydrogenase 1A1 in osteosarcoma lung metastases.

One-third of pediatric patients with osteosarcoma (OS) develop lung metastases (LM), which is the primary predictor of mortality. While current treatments of patients with localized bone disease have been successful in producing 5-year survival rates of 65-70%, patients with LM experience poor survival rates of only 19-30%. Unacceptably, this situation that has remained unchanged for 30 years. Thus, there is an urgent need to elucidate the mechanisms of metastatic spread in OS and to identify targetable molecular pathways that enable more effective treatments for patients with LM. We aimed to identify OS-specific gene alterations using RNA-sequencing of extremity and LM human tissues. Samples of extremity and LM tumors, including 4 matched sets, were obtained from patients with OS. Our data demonstrate aberrant regulation of the androgen receptor (AR) pathway in LM and predicts aldehyde dehydrogenase 1A1 (ALDH1A1) as a downstream target. Identification of AR pathway upregulation in human LM tissue samples may provide a target for novel therapeutics for patients with LM resistant to conventional chemotherapy.

Grade 2, 3 and Dedifferentiated Chondrosarcomas: A Comparative Study of Isocitrate Dehydrogenase-Mutant and Wild-Type Tumors with Implications for Prognosis and Therapy.

BACKGROUND: Grade 2 and 3 and dedifferentiated chondrosarcomas (CS) are frequently associated with isocitrate dehydrogenase (IDH) mutations and often exhibit a poor clinical outcome. Treatment is limited mainly to surgery. Defining IDH status (wild type (WT) and mutant) and the associated transcriptome may prove useful in determining other therapeutic options in these neoplasms. METHODS: Formalin-fixed paraffin-embedded material from 69 primary and recurrent grade 2, 3 and dedifferentiated CS was obtained. DNA sequencing for IDH1 and IDH2 mutations (n = 47) and RNA sequencing via Nextseq 2000 (n = 14) were performed. Differentially expressed genes (DEGs) were identified and used to predict aberrant biological pathways with Ingenuity Pathway Analysis (IPA) software (Qiagen). Gene Set Enrichment Analyses (GSEA) using subsets C3, C5 and C7 were performed. Differentially expressed genes were validated by immunohistochemistry. Outcome analysis was performed using the Wilcoxon test. RESULTS: A set of 69 CS (28 females, 41 males), average age 65, distributed among femur, pelvis, humerus, and chest wall were identified from available clinical material. After further selection based on available IDH status, we evaluated 15 IDH WT and 32 IDH mutant tumors as part of this dataset. Out of 15 IDH WT tumors, 7 involved the chest wall/scapula, while 1 of 32 mutants arose in the scapula. There were far more genes overexpressed in IDH WT tumors compared to IDH mutant tumors. Furthermore, IDH WT and IDH mutant tumors were transcriptomically distinct in the IPA and GSEA, with IDH mutant tumors showing increased activity in methylation pathways and endochondral ossification, while IDH WT tumors showed more activity in normal matrix development pathways. Validation immunohistochemistry demonstrated expression of WT1 and AR in IDH WT tumors, but not in IDH mutants. SATB2 was expressed in IDH mutant tumors and not in WT tumors. Outcome analysis revealed differences in overall survival between mutant and WT tumors (p = 0.04), dedifferentiated mutant and higher-grade (2, 3) mutant tumors (p = 0.03), and dedifferentiated mutant and higher-grade (2, 3) WT tumors (p = 0.03). The longest survival times were observed in patients with higher-grade WT tumors, while patients with dedifferentiated mutant tumors showed the lowest survival. Generally, patients with IDH WT tumors displayed longer survival in both the higher-grade and dedifferentiated groups. CONCLUSIONS: Grade 2, 3 and dedifferentiated chondrosarcomas are further characterized by IDH status, which in turn informs transcriptomic phenotype and overall survival. The transcriptome is distinct depending on IDH status, and implies different treatment targets.

Immunosurveillance shapes the emergence of neo-epitope landscapes of sarcomas, revealing prime targets for immunotherapy.

T cells recognize tumor-derived mutated peptides presented on MHC by tumors. The recognition of these neo-epitopes leads to rejection of tumors, an event that is critical for successful cancer immunosurveillance. Determination of tumor-rejecting neo-epitopes in human tumors has proved difficult, though recently developed systems approaches are becoming increasingly useful at evaluating their immunogenicity. We have used the differential aggretope index to determine the neo-epitope burden of sarcomas and observed a conspicuously titrated antigenic landscape, ranging from the highly antigenic osteosarcomas to the low antigenic leiomyosarcomas and liposarcomas. We showed that the antigenic landscape of the tumors inversely reflected the historical T cell responses in the tumor-bearing patients. We predicted that highly antigenic tumors with poor antitumor T cell responses, such as osteosarcomas, would be responsive to T cell-based immunotherapy regimens and demonstrated this in a murine osteosarcoma model. Our study presents a potentially novel pipeline for determining antigenicity of human tumors, provides an accurate predictor of potential neo-epitopes, and will be an important indicator of which cancers to target with T cell-enhancing immunotherapy.

Light-sheet autofluorescence lifetime imaging with a single-photon avalanche diode array.

Significance: Fluorescence lifetime imaging microscopy (FLIM) of the metabolic co-enzyme nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] is a popular method to monitor single-cell metabolism within unperturbed, living 3D systems. However, FLIM of NAD(P)H has not been performed in a light-sheet geometry, which is advantageous for rapid imaging of cells within live 3D samples. Aim: We aim to design, validate, and demonstrate a proof-of-concept light-sheet system for NAD(P)H FLIM. Approach: A single-photon avalanche diode camera was integrated into a light-sheet microscope to achieve optical sectioning and limit out-of-focus contributions for NAD(P)H FLIM of single cells. Results: An NAD(P)H light-sheet FLIM system was built and validated with fluorescence lifetime standards and with time-course imaging of metabolic perturbations in pancreas cancer cells with 10 s integration times. NAD(P)H light-sheet FLIM in vivo was demonstrated with live neutrophil imaging in a larval zebrafish tail wound also with 10 s integration times. Finally, the theoretical and practical imaging speeds for NAD(P)H FLIM were compared across laser scanning and light-sheet geometries, indicating a 30× to 6× acquisition speed advantage for the light sheet compared to the laser scanning geometry. Conclusions: FLIM of NAD(P)H is feasible in a light-sheet geometry and is attractive for 3D live cell imaging applications, such as monitoring immune cell metabolism and migration within an organism.

Antioxidant 1 copper chaperone gene expression and copper levels in dog osteosarcoma patients.

Twenty-four dogs with OS underwent limb amputation. Serum, OS tumour, and normal bone were harvested at time of surgery. RNA was extracted and gene expression was performed using quantitative polymerase chain reaction (qPCR). Tissue and blood copper concentrations were also determined with spectrophotometry. Compared to bone, tumour samples had significantly higher expressions of antioxidant 1 copper chaperone (ATOX1, p = .0003). OS tumour copper levels were significantly higher than that of serum (p < .010) and bone (p = .038). Similar to our previous observations in mouse and human OS, dog OS demonstrates overexpression of genes that regulate copper metabolism (ATOX1), and subsequent copper levels. Dogs with OS may provide a robust comparative oncology platform for the further study of these factors, as well as potential pharmacologic interventions.

p38MAPKα Stromal Reprogramming Sensitizes Metastatic Breast Cancer to Immunotherapy.

Metastatic breast cancer is an intractable disease that responds poorly to immunotherapy. We show that p38MAPKα inhibition (p38i) limits tumor growth by reprogramming the metastatic tumor microenvironment in a CD4+ T cell-, IFNγ-, and macrophage-dependent manner. To identify targets that further increased p38i efficacy, we utilized a stromal labeling approach and single-cell RNA sequencing. Thus, we combined p38i and an OX40 agonist that synergistically reduced metastatic growth and increased overall survival. Intriguingly, patients with a p38i metastatic stromal signature had better overall survival that was further improved by the presence of an increased mutational load, leading us to ask if our approach would be effective in antigenic breast cancer. The combination of p38i, anti-OX40, and cytotoxic T-cell engagement cured mice of metastatic disease and produced long-term immunologic memory. Our findings demonstrate that a detailed understanding of the stromal compartment can be used to design effective antimetastatic therapies. SIGNIFICANCE: Immunotherapy is rarely effective in breast cancer. We dissected the metastatic tumor stroma, which revealed a novel therapeutic approach that targets the stromal p38MAPK pathway and creates an opportunity to unleash an immunologic response. Our work underscores the importance of understanding the tumor stromal compartment in therapeutic design. This article is highlighted in the In This Issue feature, p. 1275.

T-CLEARE: A Pilot Community-Driven Tissue-Clearing Protocol Repository.

Selecting and implementing a tissue-clearing protocol is challenging. Established more than 100 years ago, tissue clearing is still a rapidly evolving field of research. There are currently many published protocols to choose from, and each performs better or worse across a range of key evaluation factors (e.g., speed, cost, tissue stability, fluorescence quenching). Additionally, tissue-clearing protocols are often optimized for specific experimental contexts, and applying an existing protocol to a new problem can require a lengthy period of adaptation by trial and error. Although the primary literature and review articles provide a useful starting point for optimization, there is growing recognition that many articles do not provide sufficient detail to replicate or reproduce experimental results. To help address this issue, we have developed a novel, freely available repository of tissue-clearing protocols named T-CLEARE (Tissue CLEAring protocol REpository; https://doryworkspace.org/doryviz). T-CLEARE incorporates community responses to an open survey designed to capture details not commonly found in the scientific literature, including modifications to published protocols required for specific use cases and instances when tissue-clearing protocols did not perform well (negative results). The goal of T-CLEARE is to provide a forum for the community to share evaluations and modifications of tissue-clearing protocols for various tissue types and potentially identify best-in-class methods for a given application.

Light sheet autofluorescence lifetime imaging with a single photon avalanche diode array.

Single photon avalanche diode (SPAD) array sensors can increase the imaging speed for fluorescence lifetime imaging microscopy (FLIM) by transitioning from laser scanning to widefield geometries. While a SPAD camera in epi-fluorescence geometry enables widefield FLIM of fluorescently labeled samples, label-free imaging of single-cell autofluorescence is not feasible in an epi-fluorescence geometry because background fluorescence from out-of-focus features masks weak cell autofluorescence and biases lifetime measurements. Here, we address this problem by integrating the SPAD camera in a light sheet illumination geometry to achieve optical sectioning and limit out-of-focus contributions, enabling fast label-free FLIM of single-cell NAD(P)H autofluorescence. The feasibility of this NAD(P)H light sheet FLIM system was confirmed with time-course imaging of metabolic perturbations in pancreas cancer cells with 10 s integration times, and in vivo NAD(P)H light sheet FLIM was demonstrated with live neutrophil imaging in a zebrafish tail wound, also with 10 s integration times. Finally, the theoretical and practical imaging speeds for NAD(P)H FLIM were compared across laser scanning and light sheet geometries, indicating a 30X to 6X frame rate advantage for the light sheet compared to the laser scanning geometry. This light sheet system provides faster frame rates for 3D NAD(P)H FLIM for live cell imaging applications such as monitoring single cell metabolism and immune cell migration throughout an entire living organism.

Intraoperative Evaluation of Soft Tissue Sarcoma Surgical Margins with Indocyanine Green Fluorescence Imaging.

Soft tissue sarcomas (STS) are rare malignant tumors often associated with poor outcomes and high local recurrence rates. Current tools for intraoperative and definitive margin assessment include intraoperative frozen section and permanent pathology, respectively. Indocyanine green dye (ICG) is a historically safe fluorophore dye that has demonstrated efficacy for intraoperative margin assessment in the surgical management of both breast and gastrointestinal cancers. The utility of ICG in the surgical management of sarcoma surgery has primarily been studied in pre-clinical mouse models and warrants further investigation as a potential adjunct to achieving negative margins. This study is a prospective, non-randomized clinical study conducted on patients with confirmed or suspected STS. Patients younger than 18 years, with a prior adverse reaction to iodine or fluorescein, or with renal disease were excluded from the study. Intravenous ICG was infused approximately three hours prior to surgery at a dosage of 2.0-2.5 mg/kg, and following tumor resection, the excised tumor and tumor bed were imaged for fluorescence intensity. When scanning the tumor bed, a threshold of 77% calibrated to the region of maximum intensity in the resected tumor was defined as a positive ICG margin, according to published protocols from the breast cancer literature. ICG results were then compared with the surgeon's clinical impression of margin status and permanent pathology results. Out of 26 subjects recruited for the original study, 18 soft tissue sarcomas (STS) were included for analysis. Three subjects were excluded for having bone sarcomas, and five subjects were excluded due to final pathology, which was ultimately inconsistent with sarcoma. The average age of patients was 64.1 years old (range: 28-83), with an average ICG dose of 201.8 mg. In 56% (10/18) of patients, ICG margins were consistent with the permanent pathology margins, with 89% specificity. The use of ICG as an intraoperative adjunct to obtaining negative margins in soft tissue sarcoma surgery is promising. However, studies with larger sample sizes are warranted to further delineate the accuracy, optimal dosage, timing, and types of sarcoma in which this diagnostic tool may be most useful.

Postoperative infection and bone sarcoma survival: systematic review of the role of infection in bone sarcoma prognosis.

Background: Osteosarcoma (OS) and chondrosarcoma (CS) are primary bone malignancies whose prognoses have stagnated despite advancements in surgical management, chemotherapy, radiation therapy, and immunotherapy. The role of the immune system in generating anti-cancer physiologic responses is critical to prognosis. Prior studies have explored if immune system activation via infection enhances survival in bone sarcomas without a clear consensus. Methods: This study sought to (I) retrospectively examine the effect of postoperative infection on survival in OS and CS and (II) systematically review the effect of postoperative infection on survival in primary bone malignancies. We performed a retrospective case-control study of 192 patients treated between 1/2000-12/2015 at a single academic sarcoma referral center. Patients with OS or CS undergoing operative resection were included. Eligible patients were grouped by presence of metastasis, and survival was compared between patients with or without postoperative infection. Furthermore, we performed a systematic review following Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines investigating the effect of infection on primary bone malignancy survival. Risk of bias assessment was performed utilizing the ROBINS-I (Risk of Bias in Non-randomized Studies-of Interventions) assessment tool. All presented studies included author information, study population, and overall or disease-free survival results. Results: One hundred and four patients were included, with 85 without infection (26 metastatic, 59 non-metastatic) and 19 with infection (10 metastatic, 9 non-metastatic). Five-year survival was greatest in patients without metastasis with a postoperative infection (100%), followed by patients without metastasis who were infection-free (80%). Five-year survival was comparatively lower in patients with metastasis who were infection-free (35%) and lowest in patients with metastasis with a postoperative infection (20%). No significant differences were present (P=0.17) on log-rank analysis. Our systematic review collected six studies exploring the impact of infection on primary bone malignancy survival, with two studies reporting significant findings of infection improving survival. Limitations of this review included risk of bias due to confounding, inconsistency comparing outcomes, and differences in patient populations. Conclusions: This retrospective study and systematic review suggests postoperative infection may play a role in modulating immune response to malignancy. Understanding the synergy between anti-pathogen and anti-cancer responses warrants further investigation as an alternative method of targeted cancer treatment.

Patient-Derived Spheroid Culture Models Are Better Than Monolayer Models in Chondrosarcoma Research.

Purpose: Chondrosarcoma (CSA) are mesenchymal tissue-derived bone tumors. CSA mainly occurs in older people. CSA has demonstrated resistance to chemotherapy and radiation; complete surgical removal with negative margins is the only treatment option. In the case of metastatic CSA, the chance of survival is meager. Since the conventional two-dimensional cell culture models failed to retain tumor characteristics, developing preclinical models mimicking the disease with the highest fidelity is paramount for personalized treatments. Methods: In this study, we established spherical cultured cells as new models for CSA. First, we demonstrated that CSA cells could form spheroids when cultured in ultra-low attachment plates. Next, tissue samples from CSA patients were collected and processed into primary cells, which were subsequently cultured as primary spheroids. The growth rate of primary spheroids was monitored and the histology of mature spheroids were characterized. These primary spheroids were used in drug susceptibility studies where traditional doxorubicin therapy and our novel disulfiram-copper therapy were tested. Results: Compared with conventional monolayer cultures, spheroids better recapitulated the features of the in vivo tumor in the aspect of the formation of extracellular matrix. In the drug susceptibility study, spheroids demonstrated high resistance to the classic therapies, suggesting that monolayer cultures may give false positive results. Therefore, using spheroids for drug research and development in the CSA field should provide more accurate results. Conclusion: In summary, our study of primary CSA spheroids brought new insight into their chemoresistance and demonstrated its potential for personalized treatment of CSA in clinical medicine.

BRCA1-associated RING domain-1 (BARD1) loss and GBP1 expression enhance sensitivity to DNA damage in Ewing sarcoma.

Ewing sarcoma is a fusion oncoprotein-driven primary bone tumor. A subset of patients (~10%) with Ewing sarcoma are known to harbor germline variants in a growing number of genes involved in DNA damage repair. We recently reported our discovery of a germline mutation in the DNA damage repair protein BARD1 (BRCA1-associated RING domain-1) in a patient with Ewing sarcoma. BARD1 is recruited to the site of DNA double stranded breaks via the poly(ADP-ribose) polymerase (PARP) protein and plays a critical role in DNA damage response pathways including homologous recombination. We thus questioned the impact of BARD1 loss on Ewing cell sensitivity to DNA damage and the Ewing sarcoma transcriptome. We demonstrate that PSaRC318 cells, a novel patient-derived cell line harboring a pathogenic BARD1 variant, are sensitive to PARP inhibition and by testing the effect of BARD1 depletion in additional Ewing sarcoma cell lines, we confirm that BARD1 loss enhances cell sensitivity to PARP inhibition plus radiation. Additionally, RNA-seq analysis revealed that loss of BARD1 results in the upregulation of GBP1 (guanylate-binding protein 1), a protein whose expression is associated with variable response to therapy depending on the adult carcinoma subtype examined. Here, we demonstrate that GBP1 contributes to the enhanced sensitivity of BARD1 deficient Ewing cells to DNA damage. Together, our findings demonstrate the impact of loss-of function mutations in DNA damage repair genes, such as BARD1, on Ewing sarcoma treatment response.

Ewing Sarcoma and Osteosarcoma Have Distinct Immune Signatures and Intercellular Communication Networks.

PURPOSE: Ewing sarcoma and osteosarcoma are primary bone sarcomas occurring most commonly in adolescents. Metastatic and relapsed disease are associated with dismal prognosis. Although effective for some soft tissue sarcomas, current immunotherapeutic approaches for the treatment of bone sarcomas have been largely ineffective, necessitating a deeper understanding of bone sarcoma immunobiology. EXPERIMENTAL DESIGN: Multiplex immunofluorescence analysis of immune infiltration in relapsed versus primary disease was conducted. To better understand immune states and drivers of immune infiltration, especially during disease progression, we performed single-cell RNA sequencing (scRNAseq) of immune populations from paired blood and bone sarcoma tumor samples. RESULTS: Our multiplex immunofluorescence analysis revealed increased immune infiltration in relapsed versus primary disease in both Ewing sarcoma and osteosarcoma. scRNAseq analyses revealed terminally exhausted CD8+ T cells expressing co-inhibitory receptors in osteosarcoma and an effector T-cell subpopulation in Ewing sarcoma. In addition, distinct subsets of CD14+CD16+ macrophages were present in Ewing sarcoma and osteosarcoma. To determine pathways driving tumor immune infiltration, we conducted intercellular communication analyses and uncovered shared mechanisms of immune infiltration driven by CD14+CD16+ macrophages and unique pathways of immune infiltration driven by CXCL10 and CXCL12 in osteosarcoma. CONCLUSIONS: Our study provides preclinical rationale for future investigation of specific immunotherapeutic targets upon relapse and provides an invaluable resource of immunologic data from bone sarcomas.

A Druggable Rheostat for Ewing Sarcoma?

The posterior HOXD enhancer is an EWSR1::FLI1-dependent regulator of HOXD13 expression in Ewing sarcoma. HOXD13 expression promotes a mesenchymal cell state. Through antagonistic transcriptional programs, EWSR1::FLI1 and HOXD13 serve as master regulators of Ewing cell plasticity. Targeting Ewing cells as they exist in/transition between mesenchymal states is a priority. See related article by Apfelbaum et al., p. 4466.

Standard metadata for 3D microscopy.

Recent advances in fluorescence microscopy techniques and tissue clearing, labeling, and staining provide unprecedented opportunities to investigate brain structure and function. These experiments' images make it possible to catalog brain cell types and define their location, morphology, and connectivity in a native context, leading to a better understanding of normal development and disease etiology. Consistent annotation of metadata is needed to provide the context necessary to understand, reuse, and integrate these data. This report describes an effort to establish metadata standards for three-dimensional (3D) microscopy datasets for use by the Brain Research through Advancing Innovative Neurotechnologies® (BRAIN) Initiative and the neuroscience research community. These standards were built on existing efforts and developed with input from the brain microscopy community to promote adoption. The resulting 3D Microscopy Metadata Standards (3D-MMS) includes 91 fields organized into seven categories: Contributors, Funders, Publication, Instrument, Dataset, Specimen, and Image. Adoption of these metadata standards will ensure that investigators receive credit for their work, promote data reuse, facilitate downstream analysis of shared data, and encourage collaboration.

Spatial transcriptomics reveals antiparasitic targets associated with essential behaviors in the human parasite Brugia malayi.

Lymphatic filariasis (LF) is a chronic debilitating neglected tropical disease (NTD) caused by mosquito-transmitted nematodes that afflicts over 60 million people. Control of LF relies on routine mass drug administration with antiparasitics that clear circulating larval parasites but are ineffective against adults. The development of effective adulticides is hampered by a poor understanding of the processes and tissues driving parasite survival in the host. The adult filariae head region contains essential tissues that control parasite feeding, sensory, secretory, and reproductive behaviors, which express promising molecular substrates for the development of antifilarial drugs, vaccines, and diagnostics. We have adapted spatial transcriptomic approaches to map gene expression patterns across these prioritized but historically intractable head tissues. Spatial and tissue-resolved data reveal distinct biases in the origins of known drug targets and secreted antigens. These data were used to identify potential new drug and vaccine targets, including putative hidden antigens expressed in the alimentary canal, and to spatially associate receptor subunits belonging to druggable families. Spatial transcriptomic approaches provide a powerful resource to aid gene function inference and seed antiparasitic discovery pipelines across helminths of relevance to human and animal health.

ALDH1A1 Gene Expression and Cellular Copper Levels between Low and Highly Metastatic Osteosarcoma Provide a Case for Novel Repurposing with Disulfiram and Copper.

Aldehyde dehydrogenase 1A1 (ALDH) is a cancer stem cell marker highly expressed in metastatic cells. Disulfiram (Dis) is an FDA-approved antialcoholism drug that inhibits ALDH and has been studied as a candidate for drug repurposing in multiple neoplasia. Dis cytotoxicity in cancer cells has been shown to be copper-dependent, in part due to Dis's ability to function as a bivalent metal ion chelator of copper (Cu). The objectives of this research were to test ALDH expression levels and Cu concentrations in sarcoma patient tumors and human osteosarcoma (OS) cell lines with differing metastatic phenotypes. We also sought to evaluate Dis + Cu combination therapy in human OS cells. Intracellular Cu was inversely proportional to the metastatic phenotype in human OS cell lines (SaOS2 > LM2 > LM7). Nonmetastatic human sarcoma tumors demonstrated increased Cu concentrations compared with metastatic tumors. qPCR demonstrated that ALDH expression was significantly increased in highly metastatic LM2 and LM7 human OS cell lines compared with low metastatic SaOS2. Tumor cells from sarcoma patients with metastatic disease displayed significantly increased ALDH expression compared with tumor cells from patients without metastatic disease. Serum Cu concentration in canine OS versus normal canine patients demonstrated similar trends. Dis demonstrated selective cytotoxicity compared with human multipotential stromal cells (MSCs): Dis-treated OS cells demonstrated increased apoptosis, whereas MSCs did not. CuCl2 combined with Dis and low-dose doxorubicin resulted in a superior cytotoxic effect in both SaOS2 and LM7 cell lines. In summary, ALDH gene expression and Cu levels are altered between low and highly metastatic human OS cells, canine samples, and patient tumors. Our findings support the feasibility of a repurposed drug strategy for Dis and Cu in combination with low-dose anthracycline to specifically target metastatic OS cells.