New biological insights into osteosarcoma-lessons from single cell sequencing studies.

Introduction: Osteosarcoma (OSA) is the most common malignant bone tumor in children and dogs. Despite aggressive treatment, no improvement in survival times has been achieved in the past 15 years with 40% of children and 90% of dogs still dying from disease. MicroRNAs (miRs) are non-protein coding RNAs that have been implicated as having a fundamental role in cancer. We analyzed primary canine OSA tumors and canine osteoblasts for miR expression and found miR-9 to be highly expressed in OSA tumors and cell lines as compared to normal osteoblasts. We hypothesize that miR-9 overexpression generates a pattern of gene dysregulation that contributes to the pathogenesis of OSA. Methodology: Canine bone marrow derived stem cells were differentiated in vitro into osteoblasts and expression of bone-specific markers (ALP, OP, BMP-2) was detected by immunocytochemistry or RT-PCR. MiR expression profiling was performed on 48 primary canine OSA tumors and 3 osteoblast cultures using the NanoString nCounter human microRNA Expression Assay. Taqman miRNA assays were used to measure miR-9 expression in primary canine OSA tumors, OSA cell lines, primary osteoblast cultures, and commercially available osteoblast cell lines. Canine OSA16 and osteoblast cell lines were transduced with lentiviral pre-miR-9 or empty control vector constructs and cells were evaluated for differences in proliferative capacity, apoptosis, and the ability to migrate through Matrigel. Results and Conclusions: We identified 84 miRs (p ≤ 0.01) differentially expressed in primary canine OSA tumors compared to canine osteoblasts. MiR-9 was highly expressed in primary OSA tumors and cell lines as compared to normal osteoblast cell lines or primary cultures. Canine OSA16 and normal osteoblast cell lines, which express low levels of miR-9 were transduced with miR-9 lentiviral constructs resulting in high levels of miR-9 expression. Overexpression of miR-9 did not effect cell proliferation or apoptosis, but enhanced invasion through matrigel in both malignant OSA cells and normal osteoblasts. Our findings suggest that miR-9 may play an important role in regulating OSA cell invasion and suggest that overexpression of miR-9 in vivo may promote metastasis in canine OSA. Transcriptional and proteomic profiling of osteoblasts overexpressing miR-9 is underway to define key differences in mRNA and protein expression and phosphorylation that may contribute to the metastatic phenotype in canine OSA. This work will provide significant new data regarding the impact of miR-9 on molecular pathways in OSA, thereby laying the foundation for the development and testing of novel therapeutics in both children and dogs affected by this disease. Citation Format: Joelle M. Fenger, Jason I. Couto, Misty D. Bear, Stefano Volinia, Jaime F. Modiano, Matthew Breen, Cheryl A. London, William C. Kisseberth. Characterization of miR-9 expression and activation in canine osteosarcoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 540. doi:10.1158/1538-7445.AM2014-540

Background: Osteosarcoma (OS) is the most common bone tumor in children and dogs; however, no substantial improvement in clinical outcome has occurred in either species over the past 30 years. MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression and play a fundamental role in cancer. Given that canine OS is a well-validated spontaneous large animal model of the human disease, the purpose of this study was to characterize the impact of miR-34a expression in canine OS tumor lines to better understand its potential role in the biology of this disease. Methods: Real-time PCR was used to assess miR-34a expression in primary canine OS tumors, canine OS cell lines, and normal canine osteoblasts. Canine OSA8 and Abrams cell lines were stably transduced with empty vector or pre-miR-34a lentiviral constructs to determine the consequences of miR-34a on cell proliferation, cell cycle distribution, invasion, and migration. Gene expression profiling of canine OSA8 cells with enforced miR-34a expression was performed using RNA sequencing and changes in mRNA expression were validated using real-time PCR. Results: Real-time PCR demonstrated that miR-34a expression levels were significantly decreased in primary canine OS tumors and canine OS cell lines as compared to normal canine osteoblasts. In canine OS cell lines stably transduced with empty vector or pre-miR-34a lentiviral constructs, overexpression of miR-34a inhibited cellular invasion and migration but had no effect on cell proliferation or cell cycle distribution. Transcriptional profiling of canine OSA8 cells possessing enforced miR-34a expression demonstrated dysregulation of numerous genes, including significant downregulation of multiple putative targets of miR-34a. Moreover, gene ontology analysis of downregulated miR-34a target genes showed enrichment of several biologic processes related to cell invasion and motility. Lastly, we validated changes in miR-34a target gene expression, including decreased expression of KLF4, SEM3A, and VEGFA transcripts in canine OS cells overexpressing miR-34a. Concordant with these data, primary canine OS tumor tissues demonstrated increased expression levels of putative miR-34a target genes. Conclusions: Our data demonstrate that miR-34a expression is significantly decreased in primary canine OS tumor tissues and canine OS cell lines compared to normal canine osteoblasts. These results are concordant with data generated in human OS tumors, suggesting that loss of miR-34a may be fundamental to the disease process in both species. Our data demonstrate that miR-34a contributes to invasion and migration in canine OS cells and suggest that loss of miR-34a may promote a pattern of gene expression contributing to the metastatic phenotype in canine OS. As such, miR-34a may represent a novel target for therapeutic intervention in OS. Citation Format: Cecilia M. Lopez, Peter Y. Yu, Xiaoli Zhang, Ayse Selen Yilmaz, Cheryl A. London, Joelle M. Fenger. MiR-34a regulates the invasive capacity of canine osteosarcoma cell lines: A comparative oncology study [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr A14.

Pulmonary metastasis is the leading cause of death in osteosarcoma (OS), the most common malignant bone tumor in children and young adults. The molecular mechanisms that regulate the formation of metastases in OS remain mostly unknown. A subset of cancer stem cells (CSCs) with metastatic potential has been identified in breast, colon and pancreatic cancers. Therefore, we hypothesize that CSCs drive the metastatic process in OS by their ability to migrate, adapt to a different microenvironment, and subsequently generate distal metastatic lesions in the lungs. Using a novel mutant p53 mouse model of metastatic OS, which has been developed in Dr. Jason Yustein's laboratory, we have identified putative OS CSCs in primary bone tumors and metastatic lesions. Cancer stem cells have been cultured from solid tumors growing as sphere-like structures under serum-free conditions. This method has been validated as a surrogate of their self-renewal capacity. We have observed that murine tumor cells obtained from both primary bone tumors and pulmonary nodules developed sarcospheres. Notably, sarcophere-forming efficiency was significantly increased (2-3 times) in tumor cells obtained from metastatic lesions. Aldehyde dehydrogenase 1 (ALDH1) activity has been used as a functional stem cell marker to isolate CSCs in numerous solid tumors. We have found that both primary bone tumors and metastatic pulmonary nodules contain ALDH Hi (stem-like cells). The high expression of ALDH1 in murine OS tumors ranged from 0.7 to 17%. Further analysis of derived cell lines generated from primary OS tumor, circulating tumor cells (CTCs) and metastatic tumors showed that distal metastases have 4-5 times more ALDH Hi cells than CTCs and up to 10 times more ALDH Hi cells than the primary bone tumors. The isolation of OS CSCs by fluorescent activated cell sorting (FACS) facilitates the analysis of the molecular pathways and biological process that regulates this tumor cell compartment. Our initial data has shown that the Insulin-like Growth Factor (IGF) family members were differentially expressed between OS primary and metastatic cells. Furthermore, we are actively utilizing these mouse derived cell lines to perform orthotopic transplants in immunocompetent (syngeneic) mice aiming to better understand the biology and genetics of CSCs in OS. Subsequent studies will be performed to determine the functional significance of the unique OS CSC genetic alterations. In summary, we have identified putative metastatic OS CSCc in a mutant p53 mouse model of metastatic OS. Our preliminary data has shown that CSC content seems to be increased in metastases. Our studies using an OS mouse model that parallels the biological behavior of human disease in addition to provide freshly isolated tumor specimens constitute a strong pre-clinical model to test the effect of novel anti-cancer therapies in the CSC compartment. Citation Format: Nino Rainusso, Lyazat Kurenbekova, Lawrence Donehower, Jeffrey Rosen, Jason Yustein. Characterization of metastatic cancer stem cells in osteosarcoma. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C80.

This study was undertaken to investigate the therapeutic effect of a genetically engineered, human pre-microRNA-34a prodrug in canine osteosarcoma (OS), validating a robust preclinical model for the development of this proposed agent in humans. Although osteosarcoma is the most prevalent primary bone tumor in children and young adults, the low overall incidence presents an obstacle to timely development and testing of new agents. However, the incidence of canine OS is nearly 12 times that in humans, providing a useful preclinical model with spontaneously occurring OS. Despite multimodal therapeutic protocols, up to 40% of human and 90% of dogs will not survive long-term. Therefore, there is a clear need to identify new therapeutic agents for OS treatment. MicroRNAs (miRs) are small, noncoding RNAs that control relevant cellular pathways in tumorigenesis. MiR-34a, a downstream component of the p53 tumor suppressor, is downregulated in OS tumors and its reduced expression linked to poor therapeutic response. Recent evidence in human OS cell lines has shown that a novel genetically engineered human tRNA/miR-34a prodrug has anti-tumor effects following intracellular processing into mature miR-34a. We transfected a panel of canine OS cell lines (D17, HMPOS, Abrams and Gracie) with the human tRNA/miR-34a prodrug and evaluated the downstream effects to validate canine OS as a preclinical model. We measured miR-34a levels following transfection using quantitative real-time PCR. Proliferation was assessed via bioreductive and clonogenic assays. Western blot and immunofluorescence microscopy were used to investigate miR-34a target protein expression. Caspase 3/7 activity and TUNEL staining were used to measure apoptotic effects. In vitro properties associated with metastatic ability were assessed via transwell migration and invasion assays using Boyden chambers. We found significant increases in mature miR-34a levels following transfection, indicating that canine cells effectively process the prodrug. Increased miR-34a was associated with reduced PDGFRα protein expression and dose- and time-dependent inhibition of proliferation. Compared to control and MSA-transfected cells, miR-34a significantly reduced clonogenic cell growth, increased caspase 3/7 activity, and increased TUNEL positivity. We also found a significant inhibition of canine OS cell migration and invasion following transfection with miR-34a prodrug. Taken together, our findings demonstrate the successful internalization and processing of human miR-34a prodrug by canine OS cells. The increased miR-34a levels produce the same anti-tumor effects seen in human OS cells. These results strongly supports the use of canine OS as a preclinical model for the development of this therapy in human OS. Citation Format: Fernando Alegre Guerra, Kellie Snider, Amanda Ormonde, Ai-Ming Yu, Luke Wittenburg. Canine osteosarcoma as a platform to investigate the therapeutic potential of a genetically engineered pre-microRNA prodrug in human osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4406.

Multiomic single nucleus RNA- and ATACseq profiling reveals regulators of glioma cell state diversity. The extensive intra- and intertumoral heterogeneity observed in glioma reflects the resistance to therapy and poor prognosis observed clinically. Single-cell sequencing studies have highlighted that glioma heterogeneity reflects the co-existence of cell subpopulations with distinct cell states. Prior studies have also shown that EGFR-amplifying extrachromosomal DNA (ecDNA) elements in IDH-wild-type gliomas can contribute to heterogeneity by driving oncogene amplification through long range chromatin contacts. However, single cell studies have largely focused on analyses of transcriptional profiles, and the epigenetic mechanisms underlying the contribution of ecDNA elements to tumor cell state diversity remain poorly understood. To further our understanding of the regulatory programs that contribute to transcriptional diversity and mediate the distribution of tumor cell states, we profiled primary-recurrent tumor pairs from 18 patient samples with multiomic single-nucleus RNA- and ATACseq, resulting in 86,135 cells identified with linked chromatin accessibility and gene expression profiles. Integrative clustering of the tumor cells identified tumor cell states ranging from a stem-like to differentiated- phenotype that were also associated with differences in chromatin accessibility and inferred transcription factor binding activity. Analyses of chromatin accessibility resulted in the identification of ecDNA, and integrative clustering of ecDNA+ cells highlighted distinct cell states with increased copy number burden, oncogene amplification, and differential chromatin accessibility. These results suggest that a better understanding of extrachromosomal contributions to tumor diversity would aid in development of more efficient therapies.

MicroRNAs (miRNAs) are a class of small noncoding RNAs that are implicated in various important biological processes by regulation of gene expression. Aberrant miRNA expression is suggested to be associated with various human disorders including cancer. In this study, we studied if miRNAs can be used as biomarkers in osteosarcoma (OS). OS is the most common malignant bone tumor in children and young adults. Despite the use of surgery and multi-agent chemotherapy, OS patients who respond poorly towards chemotherapy or develop relapses have a dismal outcome. Thus, it is of clinical significance if a biomarker approach can be developed to monitor tumor burden and detect early relapses in the patients, so that they can be treated as early as possible to improve the survival. To achieve this goal, we performed miRNA profiling on a cohort of OS plasma samples collected from the Texas Children's Hospital. miR-21 and other miRNAs were identified to be elevated in OS patients relative to a group of healthy controls. Circulating miR-21 has been detected in other types of cancer and in the tumor it is known to negatively regulate tumor suppressor genes involved in proliferation, apoptosis and invasion; however, its role in OS is still unclear. Hence, we first validated that miR-21 was elevated in a set of independent osteosarcoma plasma samples but not in the control samples. Using serial plasma samples from a group of patients, we found that the circulating miR-21 level was lower in the posttreatment plasma samples than in the pretreatment samples. These results suggest that circulating miR-21 may correlate with tumor burden of the patients. In addition, miR-21 was expressed in many OS tumors as detected by in situ hybridization on an OS tissue microarray. When tested on two OS cell lines, miR-21was significantly increased in the culture media between 24 and 48 hours after plating in a cell number dependent manner, suggesting that miR-21 could be released from the tumor cells. Taken together, our results suggest that miR-21 is expressed in OS and its level in plasma may reflect tumor burden in OS patients. Citation Format: Manjula Nakka, Yiting Li, Colin McGee, Ching Lau, Tsz Kwong Man. Characterization of miR-21 as a potential circulating biomarker for osteosarcoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5228. doi:10.1158/1538-7445.AM2014-5228

Osteosarcoma (OS) is the most common primary malignant bone tumor in children and adolescents, characterized by aggressive behavior and poor prognosis. Despite advances in multimodal therapies, outcomes for patients with metastatic or recurrent OS remain poor. Non-SMC Condensin II Complex Subunit D3 (NCAPD3) is a core component of the condensin II complex involved in chromosome condensation and segregation; however, its role in OS progression and the tumor immune microenvironment remains unclear. Publicly available transcriptomic datasets were analyzed to evaluate NCAPD3 expression and prognostic relevance in OS. Using single-cell RNA sequencing (scRNA-seq; n = 6), we characterized tumor cell heterogeneity and NCAPD3-expressing subsets within the osteosarcoma tumor microenvironment. In vitro, NCAPD3 knockdown was performed in RAW264.7 macrophages, primary murine macrophages, and THP-1-derived macrophages to assess changes in macrophage polarization markers at the transcriptional and protein levels. Functional assays, including conditioned-media experiments, evaluated tumor-macrophage crosstalk. The effects of NCAPD3 on osteosarcoma cell proliferation, migration, and tumor growth were assessed in vitro and in vivo. Pathway enrichment analyses were conducted in NCAPD3⁺ macrophages and tumor cells, and in silico drug sensitivity prediction was performed as an exploratory analysis. NCAPD3 was significantly overexpressed in OS tissues and associated with poorer overall survival. scRNA-seq analysis identified distinct tumor cell subsets and a population of NCAPD3⁺ macrophages characterized by reduced inflammatory activity. NCAPD3 knockdown in macrophages consistently upregulated pro-inflammatory markers and downregulated anti-inflammatory markers, indicating a shift toward a pro-inflammatory phenotype. Conditioned-media experiments demonstrated that NCAPD3-dependent tumor-macrophage crosstalk regulates osteosarcoma cell proliferation and migration. Pathway analyses revealed downregulation of IL2/STAT5 and IL6/JAK/STAT3 signaling in NCAPD3⁺ macrophages, while NCAPD3⁺ tumor cells exhibited reduced apoptotic signaling and enhanced DNA repair and PI3K/AKT/mTOR pathway activity. Functional assays confirmed that NCAPD3 promotes osteosarcoma cell growth and tumor progression in vitro and in vivo. NCAPD3 promotes osteosarcoma progression through coordinated effects on tumor cell proliferation and suppression of macrophage-mediated inflammatory responses. These findings identify NCAPD3 as a context-dependent regulator of tumor-immune interactions in OS and support its potential as a prognostic biomarker and therapeutic target.

Purposes: We would like to inform the characteristics of recurred osteo-fibrous dysplasia (OFD), and also the possible use of vascularized composite autograft and allografts (VCA) combined with live fibular graft (LFG) for the treatment of aggressive benign bone tumor and osteogenic sarcomas in children. Materials and Methods: We reviewed one boy with recurred OFD after LFG, and other four children with osteogenic sarcoma in long bone which was treated with LFG and VCA, and followed them for average 9 years (3 - 14 years). Survival of the LFG and VCA was estimated by the ISOLS Functional Radiologic Scoring System, but not with Kaplans-Meier’s scoring system because of a small series of case reports. Results: All succeed after surgeries initially, but the cause of recurrence of OFD was still unclear, and one girl with osteogenic sarcoma on distal femur died with skip and lung metastasis, 4 years after surgeries. Conclusions: The causes of recurred OFD are not found thru pathologic studies of our patient, but we believe the multifocal origin of tumor cells even on the adjacent soft tissues in OFD is one of the causes. The excised long bone tumor is recycled by pasteurization or autoclaving, or allograft, then LFG, and neo-adjuvant chemotherapy would be one of elective surgery for the treatment of malignant long bone tumors in children. The LFG into recycled autograft or allograft bone is difficult to perform simultaneously, but very effective to increase more earlier regenerative vascularities and also the stabilities of the dead bones in children.

Osteosarcoma (OS) is the most common malignant bone tumor in children and teenagers. In many cases, such as poor response to treatment or the presence of metastases at diagnosis, the survival rate of patients remains very low. Although in the literature, more and more studies are emerging on the role of Ubiquitin-Specific Proteases (USPs) in the development of many cancers, few data exist regarding OS. In this context, RNA-sequencing analysis of OS cells and mesenchymal stem cells differentiated or not differentiated into osteoblasts reveals increased expression of four USPs in OS tumor cells: USP6, USP27x, USP41 and USP43. Tissue microarray analysis of patient biopsies demonstrates the nucleic and/or cytoplasmic expression of these four USPs at the protein level. Interestingly, Kaplan–Meyer analysis shows that the expression of two USPs, USP6 and USP41, is correlated with patient survival. In vivo experiments using a preclinical OS model, finally demonstrate that PR619, a USP inhibitor able to enhance protein ubiquitination in OS cell lines, reduces primary OS tumor growth and the development of lung metastases. In this context, in vitro experiments show that PR619 decreases the viability of OS cells, mainly by inducing a caspase3/7-dependent cell apoptosis. Overall, these results demonstrate the relevance of targeting USPs in OS.

Osteosarcoma (OS) is the most common primary malignant bone tumor in children, with a particularly high incidence and high rates of metastatic disease in Hispanic and African American patients. With the goal of understanding disparities within underrepresented communities affected by pediatric OS, we recently described the epidemiology of pediatric OS within a Hispanic patient cohort. We demonstrated that malignant bone tumors represent 4.68% of all pediatric cancers in Hispanics living in Puerto Rico, with an age-adjusted incidence of 6.02 cases per million. Notably, 34.62% of these patients present localized disease while 42.3% have metastatic disease at diagnosis with a mortality rate of 2.4 per million. These statistics highlight the need for dedicated studies to address clinical and biological causes for disparities in underserved populations. The Human Epidermal Growth Factor Receptor 4 (HER4), a member of the EGFR receptor tyrosine kinase family, has been demonstrated to be upregulated in pediatric OS patients leading to decreased overall survival. This receptor regulates pathways responsible for cell survival, proliferation, and differentiation and has been implicated in tumorigenesis in various cancers. Furthermore, the EGFR family can also modulate immune cell populations, although this is largely understudied in HER4 positive tumors including aggressive pediatric osteosarcoma. The goal of our study is to evaluate HER4 expression in a cohort of Hispanic patients with pediatric osteosarcoma and determine whether HER4 expression can be predetermined by genetic ancestry. We hypothesize that underrepresented minorities including Hispanics and African Americans have higher HER4 expression, predisposing these patients to aggressive OS. By retrospective medical record review between 2009-2023, we identified 45 patients between the ages of 1-19 years diagnosed with pediatric OS. Thirty-one pediatric OS tissue blocks were deemed adequate for further studies, including biopsies, metastases, resections, and relapses from the Pathology Department of the Puerto Rico Medical Center. We performed immunohistochemistry (IHC) for HER4 expression using a validated monoclonal HER4 antibody (Invitrogen). Our results show high HER-4 expression in the majority of our Hispanic patient cohort with pediatric OS, suggesting that HER4 may play drive a crucial step in OS tumor progression and metastasis within these communities. Ongoing analysis includes evaluating HER4 expression within other ethnic groups including Hispanics in US, Caucasians, and African Americans. Furthermore, we are also evaluating the immune cell compartment in HER4+ pediatric OS by single cell sequencing and spatial transcriptomics. Understanding how genetic ancestry can lead to disparities in pediatric OS has the potential to result in effective targeted therapeutic strategies for underserved minorities. Citation Format: Rocio Krystal Rivera-Valentin, Carolyn M. Ruiz-Perez. Evaluating HER4 as a potential driver of disparities in Hispanics with pediatric osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 707.

Osteosarcoma (OS) is the most common non-hematologic primary tumor of bone in children and adults. High-dose cytotoxic chemotherapy and surgical resection have improved prognosis, with long-term survival for non-metastatic disease approaching 70%. However, most OS tumors are high grade and tend to rapidly develop pulmonary metastases. Despite clinical advances, patients with metastatic disease or relapse have a poor prognosis. Here the cell biology of OS is reviewed with a special emphasis on mouse models as well as the roles of the cell of origin and cancer stem cells. A better understanding of the molecular pathogenesis of human OS is essential for the development of improved prognostic and diagnostic markers as well as targeted therapies for both primary and metastatic OS.

Osteosarcoma (OS) is the most common non-hematologic primary tumor of bone in children and adults. High-dose cytotoxic chemotherapy and surgical resection have improved prognosis, with long-term survival for non-metastatic disease approaching 70%. However, most OS tumors are high grade and tend to rapidly develop pulmonary metastases. Despite clinical advances, patients with metastatic disease or relapse have a poor prognosis. Toward a better understanding of the molecular pathogenesis of human OS, several genetically modified OS mouse models have been developed and will be reviewed here. However, better animal models that more accurately recapitulate the natural progression of the disease are needed for the development of improved prognostic and diagnostic markers as well as targeted therapies for both primary and metastatic OS.

Bone deposition and bone resorption are ongoing dynamic processes, constituting bone remodeling. Some bone tumors, such as osteosarcoma (OS), stimulate focal bone deposition. OS is the most common primary bone tumor in children and young adults. A complex network of genes regulates bone remodeling and alterations in its expression levels can influence the genesis and progression of bone diseases, including OS. We hypothesized that the expression profiles of bone remodeling regulator genes would be correlated with OS biology and clinical features. We used real-time PCR to evaluate the mRNA levels of the tartrate-resistant acid phosphatase (ACP5), colony stimulating factor-1 (CSF1R), bone morphogenetic protein 7 (BMP7), collagen, type XI, alpha 2 (COL11A2), and protein tyrosine phosphatases zeta 1 (PTPRZ1) genes, in 30 OS tumor samples and correlated with clinical and histological data. All genes analyzed, except CSF1R, were differentially expressed when compared with normal bone expression profiles. In our results, OS patients with high levels of COL11A2 mRNA showed worse overall (p = 0.041) and event free survival (p = 0.037). Also, a trend for better overall survival was observed in patients with samples showing higher expression of BMP7 (p = 0.067). COL11A2 overexpression and BMP7 underexpression could collaborate to OS tumor growth, through its central role in bone remodeling process.

Background: Treatment resistance in solid tumors is driven by clonal evolution, with therapies selecting for resistant subpopulations. Single-cell sequencing (SCS) and phylogenetic analyses reveal branching patterns and subclonal dynamics, but no meta-analysis has synthesized their prognostic impact. This study quantifies evolutionary drivers of resistance across solid tumors, informing adaptive therapy design. Methods: PubMed, Scopus, and Web of Science (2015–2025) were searched for cohort studies or trials reporting SCS or phylogenetic data in solid tumors (e.g., breast, lung, colorectal) with resistance endpoints (e.g., progression-free survival [PFS], clonal shifts). Inclusion: human studies, ≥10 patients, evolutionary metrics (e.g., clonal diversity, variant allele frequencies), resistance defined (HR >1.5 for PFS). Exclusion: non-solid tumors, reviews. Data extracted: study design, tumor type, sample size, diversity metrics (e.g., Shannon index), and outcomes (HR for PFS/OS). Risk of bias used QUADAS-2. Random-effects models in RevMan 5.4 computed standardized mean differences (SMD) for subclonal fraction and hazard ratios (HR) with 95% CIs. Heterogeneity assessed via I2; publication bias via Egger’s test. Results: From 1,247 records, 28 studies (n=2,456 patients; 58% female, mean age 62) were included: breast (n=9), lung (n=10), colorectal (n=6), other (n=3). Most used SCS (e.g., 10x Genomics) or phylogenetic tools (e.g., PyClone). Pooled subclonal fraction was higher in resistant tumors (SMD 1.82, 95% CI 1.40–2.24; I2=70%; 22 studies; P<0.001). Pooled HR for PFS was 2.10 (95% CI 1.66–2.65; I2=64%; 18 studies), with lung tumors showing stronger effects (HR 2.42, 95% CI 1.87–3.14) than breast (HR 1.75, 95% CI 1.30–2.36). Meta-regression linked branching evolution to worse outcomes (β=0.30, P=0.03). No publication bias (Egger’s P=0.22). Sensitivity analyses excluding high-bias studies (n=3) altered HR by <5%. Conclusions: Higher subclonal diversity predicts resistance (HR>2) across solid tumors, supporting evolutionary metrics in trial designs. This first meta-analysis of phylogenetic/SCS data resolves prior inconsistencies, aligning with AACR’s focus on tumor progression dynamics. Citation Format: Noureddine SAMAI. Meta-analysis of evolutionary drivers of treatment resistance in solid tumors: Insights from phylogenetic and single-cell sequencing studies [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Cancer Evolution: The Dynamics of Progression and Persistence; 2025 Dec 4-6; Albuquerque, NM. Philadelphia (PA): AACR; Cancer Res 2025;85(23_Suppl):Abstract nr A005.

Diagnosis and longitudinal monitoring of neurological diseases are limited by the poor specificity and limited resolution of currently available techniques. Analysis of circulating cells in cerebrospinal fluid (CSF) has emerged as a promising strategy for the diagnosis, molecular characterization, and monitoring of neurological disease. In comparison to bulk sequencing analysis, single-cell sequencing studies can provide novel insights into rare cell populations and uncover heterogeneity in gene expression at a single-cell resolution, which has several implications for understanding disease pathology and treatment. Parallel development of standardized biofluid collection protocols, pre-processing strategies, reliable single-cell isolation strategies, downstream genomic analysis, and robust computational analysis is paramount for comprehensive single-cell sequencing analysis. Here we perform a comprehensive review of studies focusing on single-cell sequencing of cells in the CSF of patients with oncological or non-oncological diseases of the central nervous system.