Gonzalez-Aponte, Maria F.

Abstract Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adults with a poor prognosis of 15 months despite aggressive therapies. GBM malignancy is often driven by a high degree of inter- and intra-tumoral heterogeneity that is dynamic through time and regulated by interactions between tumor cells and their neural microenvironment. For instance, GBM tumors located near the lateral ventricles (LV) of the brain grow more aggressively and result in significantly worse patient survival, compared to tumors that grow far from the LV. This suggests that interactions between GBM and cells in the LV microenvironment may regulate tumor malignancy. Understanding the mechanisms underlying GBM malignancy near the LV is essential to determine if regulation of tumor biology is driven by the presence of healthy neural cells, and to ultimately develop therapeutic approaches that target GBM-LV interactions. Here, we tested the hypothesis that region-specific cellular identities and activation of unique transcriptional programs regulate GBM malignancy near the LV. Paired primary biopsies were collected from 14 GBM patients. Image-guided biopsies proximal and distal to the brain’s LV (LV-proximal GBM and LV-distal GBM, respectively) were processed for bulk RNA sequencing, single-nucleus RNA sequencing, and in situ spatial transcriptomic (Xenium) analyses. We found significant inter-patient and intra-tumoral transcriptional heterogeneity in GBM samples collected proximal and distal to the LV. After accounting for tumoral and patient heterogeneity, we found that proximal GBM tumors exhibit an increased immune-related gene signature, compared with tumors distal to the LV. Single-nucleus RNA sequencing and in situ spatial transcriptomic analyses showed no significant differences in the proportion of cellular states or identities between proximal and distal GBM samples. However, within each cellular identity, significant differences in gene expression were identified between proximal and distal GBM, suggesting that distinct cell-specific transcriptional pathways characterize regional differences in GBM biology. Our results suggest that GBM tumors near the LV have increased interactions with immune cells that lead to the enrichment of immune-related transcriptional pathways, compared to tumors growing far from the LV. This work will inform the development of therapies that target GBM-immune cell interactions to decrease tumor malignancy and improve individual patient outcomes. Citation Format: Maria F. Gonzalez-Aponte, Jhan C. Salazar, Aura Figueroa-Gonzalez, Emily S. Norton, Paola Suarez-Meade, Hugo Guerrero-Cazares. Integrating RNA sequencing and spatial transcriptomic analyses reveals drivers of glioblastoma malignancy near the brains’ lateral ventricles [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Brain Cancer; 2026 Mar 23-25; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2026;86(6_Suppl):Abstract nr B035.

Abstract Cancer remains one of the most complex and devastating diseases, in part because tumors are not static entities but dynamic ecosystems that evolve in real time. Over the decades, researchers have proposed two major frameworks toexplain tumor development: 1) the clonal evolution model, which emphasizes the stepwise accumulation of mutations insingle cells, and 2) the cancer stem cell model, which posits a hierarchical tumor structure driven by a small population of stem-like cells capable of self-renewal and tumor initiation. In brain tumors, as in other cancer types, the presence of cancer stem cells has been demonstrated; however, their evolutionary role in glioblastoma, the most aggressive primary brain tumor, remains poorly understood. A key unanswered question is how cancer stem cells in the brain evolve intomalignant populations and how evolutionary processes shape this transition. Since Darwin, evolutionary biologists have sought to understand how traits evolve across lineages and have developed several tools to address questions regarding how lineages, species and populations have evolved through time. In recent years there has been an increase in the use of evolutionary statistical methods to study cancer evolution. These approaches remain niche in a few tumor types (e.g., blood and lung cancer), but the rapid growth of sequencing technologies and large-scale datasets has created new opportunities to address evolutionary questions across cancer types that have been understudied (e.g., brain tumor). Researchers are now using whole-genome, exome, single-cell, spatial, and multi-omic data to reconstruct tumor histories, test adaptive hypotheses, and in few cases, quantify rates and modes of evolution within tumors and between tumor types. However, there is not yet a consolidated framework for which evolutionary analyses are most appropriate to test cancer evolution, nor accessible guidance on how to implement these approaches in oncological datasets. We synthesize evolutionary statistical methods currently used in brain cancer research and propose additional approaches for future studies. We identify more than 30 evolutionary tools developed to study trait evolution, diversification, and lineage dynamics in biological systems, of which only one has been applied to brain cancer. We argue that adapting these methods offers a powerful opportunity to investigate cancer stem cell dynamics and tumor progression in glioblastoma and other understudied cancers. By bridging evolutionary biology and cancer genomics, this work provides a roadmap for expanding the evolutionary toolkit used to study tumor evolution and for generating testable hypotheses about malignant transformation. Citation Format: Jhan C. Salazar Salazar, Sean W. McHugh, Maria F. Gonzalez-Aponte, Hugo Guerrero-Cazares. From healthy brain to cancer cells: Understanding the origins of glioblastoma through the lens of Darwinian evolution [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Brain Cancer; 2026 Mar 23-25; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2026;86(6_Suppl):Abstract nr B017.

Glioblastoma (GBM) is an aggressive brain tumor that often progresses despite resection and treatment. Timely and continuous assessment of GBM progression is critical to expedite secondary surgery or enrollment in clinical trials. However, current progression detection requires costly and specialized MRI examinations, which, in the absence of new symptoms or signs, are usually scheduled every 2–3 months. Here, we hypothesized that changes in daily activity are associated with GBM growth and disease progression. We found that wheel-running activity in GBM-bearing mice declined as tumors grew and preceded weight loss and circadian breakdown by over a week. Temozolomide treatment in the morning, but not evening, significantly reduced tumor size and restored daily locomotion in mice. In a pilot study of 6 patients with GBM wearing an actigraphy watch, wrist movement provided a feasible and continuous longitudinal indicator of daily activity with 1-minute resolution. After tumor resection and radiation, daily activity declined in 2 patients 19 and 55 days before detection of progression by MRI but did not change for the 4 patients with stable disease. These results suggest that daily activity tracking using wearable devices may serve as a real-time indicator and potential monitoring tool for GBM progression and treatment efficacy.

Abstract Glioblastoma (GBM) is the most common primary tumor in adults with a median survival of 15 months post-treatment. GBM incidence and progression depends on sex, with reports of 1.6x increased incidence and 6-month shorter overall survival in males, compared to females. Regardless of sex, the standard of care for GBM patients includes surgical resection, followed by chemotherapy and radiotherapy, and often dosing of the synthetic glucocorticoid Dexamethasone (DEX) to reduce brain edema post-treatment. Our lab recently found that DEX can suppress or promote GBM growth depending on the time of day the treatment is administered. Because glucocorticoid secretion depends on circadian time and sex, we tested whether endogenous glucocorticoids regulate sex differences in GBM growth. We transduced a murine chromosomal male or female GBM cell line (Nf1-/-DNp53 XX or XY) with a transcriptional luciferase reporter of the circadian clock gene Per2 (Nf1-/-DNp53-P2L). These cells were next treated with DEX at the daily peak or trough of Per2 expression and growth was measured 48 hours post-treatment. We found that administering DEX at peak Per2 suppresses growth in males, while administering DEX at trough Per2 promotes growth in both males and females. We then knocked-down the glucocorticoid receptor (GR) using an shRNA lentiviral construct in two GBM cell models (GL261 and LN229) and implanted WT or GR-KD cells into the basal ganglia of male and female mice. Loss of GR in GBM cells significantly suppressed GBM growth, especially in male mice, as evidenced by postmortem Ki67 staining. These results suggest that GBM growth in males may be more sensitive to endogenous glucocorticoids, and that sensitivity varies with time of day. This work will inform future personalized circadian interventions to improve GBM patient outcomes. Supported by NIH grant R01NS134885.

High-grade gliomas, like glioblastoma multiforme (GBM), are the most common malignant brain tumors in adults and are treated with the chemotherapy drug temozolomide (TMZ). In humans, a retrospective analysis of patients’ overall survival suggests that morning dosing may confer a benefit over evening dosing. Circadian variation in O6-methylguanine-DNA methyltransferase (MGMT) gene expression and promoter methylation has been implicated in increased tumor cell sensitivity to TMZ in the morning. Although patient compliance with timed oral administration of TMZ was high in a prospective trial, it is not known whether differences in daily sleep patterns of patients impact the biological time of drug administration or overall survival. Using wrist actigraphy collected from 10 high-grade glioma patients, we quantified the moment of oral TMZ delivery in terms of wall clock time and internal biological time during the months after surgical tumor resection. We found that variation of daily rhythms within and between individuals caused dosing times to vary more in their internal biological time than wall clock time so that, for example, some doses taken by patients assigned for the evening (2000 h) were closer to the patient’s internal biological morning. We conclude that wrist actigraphy provides a reliable and non-invasive estimate of personal circadian time that could improve efficacy and precision of TMZ delivery. These findings may inform personalized circadian medicine and optimized times for TMZ delivery in the clinic.