Biology subjects

Aguilera, T. A.

Publications and source records attributed to Aguilera, T. A..

4 recordsLinked to original sources

bioRxiv2025

Resolving phenotyping discordance with SPACEMAP, an integrated machine learning framework

Multiplex imaging technologies have revolutionized our ability to study cellular behavior within the tissue microenvironment. Translating this complex data into meaningful biological insights requires a unified analytical framework. To address this, we developed SPACEMAP (Spatial Phenotyping And Classification with Enhanced Multiplex Analysis Pipeline), a comprehensive Python and Qupath-based platform for multiplex imaging analysis. SPACEMAP integrates image registration, segmentation, artifact removal, tissue and zone classification, spatial feature extraction, and a consolidated phenotyping approach into a single system. A core feature of SPACEMAP is its high-fidelity phenotyping. To evaluate classification performance, we benchmarked our method RESOLVE, against three established approaches, Leiden clustering, Self-Organizing Maps, and SCIMAP revealing substantial disagreement among them. SPACEMAP overcomes this through two complementary workflows: a machine learning model trained on expert-labeled cells, and a consensus classifier that integrates high-confidence cells across methods. Here, we validated SPACEMAP on in-house colorectal cancer samples and a public dataset, demonstrating its robustness.

bioinformatics↗

bioRxiv2025

Immune stromal components impede biological effectiveness of carbon ion therapy in a preclinical model of pancreatic ductal adenocarcinoma

The tumor landscape of pancreatic ductal adenocarcinoma (PDAC) is refractory to conventional photon radiotherapy (RT) due to a fibrotic tumor microenvironment (TME) that promotes chronic hypoxia and reduced immune surveillance. The radiobiological factors unique to carbon ion radiotherapy (CIRT), such as high linear energy transfer (LET) and less dependence on oxygen, make it well-suited to overcome the PDAC TME. Here, we utilized clonal syngeneic KPC pancreatic tumor cell lines and tumors to examine this postulate and to identify underlying factors that impact the response of PDAC to CIRT. While KPC cell lines exhibited radiobiologic effectiveness (RBE) greater than 3, subcutaneous tumors in the mouse hind leg showed lower RBEs - 1.3 based on quintupling time - at a LET of 75 keV/m. Four days after CIRT, we observed widespread transcriptomic changes in the tumor immune microenvironment (TME), suggesting increased infiltration of anti-tumor immune cells, elevated expression of anti-tumor T cell cytokines, MHC class I molecules, and co-stimulatory signals. Fewer immunologic changes were observed following photon irradiation. By seven days after CIRT, tumor-supportive transcriptomic programs characterized by pro-tumor cytokines, M2 macrophages, and cancer-associated fibroblasts (CAFs) emerged, promoting resistance and limiting the durability of tumor growth delay. These findings suggest that CIRT may offer a favorable platform compared to conventional photon radiation therapy for combining with immunotherapies. Furthermore, these data highlight the risk of using in vitro survival data alone in treatment planning and indicate that underlying TME factors impact the response of PDAC in vivo.

cancer biology↗

bioRxiv2024

Defective Homologous Recombination and Genomic Instability Predict Increased Responsiveness to Carbon Ion Radiotherapy in Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is inherently resistant to conventional chemo-and radiation-therapy. However, clinical trials showed that carbon ion radiotherapy (CIRT) with concurrent gemcitabine can be effective for treating unresectable locally advanced PDAC. In this study, we aimed to determine features that could identify patients who would benefit most from CIRT. A panel of human PDAC cell lines with various genetic backgrounds was leveraged to determine whether a subset could be identified that preferentially responds to CIRT. The cell lines displayed a differential response to CIRT as compared to {gamma}-rays as measured by relative biological effectiveness (RBE) calculated at 10% survival, which ranged from 1.96 to 3.04. Increased radiosensitivity correlated with decreased DNA double strand break (DSB) repair as measured by {gamma}H2AX foci resolution. We determined that the cell lines most sensitive to CIRT are defective in the homologous recombination (HR) DSB repair pathway and/or have high genomic instability due to elevated replication stress. Next, this knowledge was utilized to assess whether the HR pathway could be targeted to potentiate CIRT in vitro. It was determined that pretreating a radioresistant PDAC cell line with the HR inhibitor, B02, resulted in a marked increase in sensitivity to CIRT when treated with high linear energy transfer (LET) radiation in the spread-out Bragg peak (74.1-89.3 keV/m) but not at the entry LET (13.0-16.4 keV/m) in vitro as opposed to that seen with a NHEJ inhibitor. These data suggest a greater therapeutic index with the combination therapy in the tumor over normal tissues based on LET distribution. These data support the notion that PDAC tumors with defects in HR and/or those with high inherent replication stress respond to CIRT without the concern for excessive normal tissue toxicity. With the advent of agents targeting HR, the difference in tumor cell response in the entry region vs within the SOBP based upon the respective LETs of those regions of the beam profile, will be superior to agents that target NHEJ.

cancer biology↗

bioRxiv2021

In vivo selection of nanobodies to target the tumor immune microenvironment for immunotherapy development

In the rapidly advancing field of synthetic biology, there is a critical need for technology to discover targeting moieties for therapeutic biologics. We developed INSPIRE-seq, an approach that utilizes a nanobody library and next-generation sequencing to identify nanobodies selected for complex environments. INSPIRE-seq enables the parallel enrichment of immune cell-binding nanobodies that penetrate the tumor microenvironment. Clone enrichment and specificity varies across immune cell subtypes in the tumor, lymph node, and spleen. INSPIRE-seq identified a dendritic cell binding clone that binds PHB2. Single-cell RNA sequencing revealed a connection with cDC1s, and immunofluorescence confirmed nanobody-PHB2 colocalization along cell membranes. Structural modeling and docking studies assisted binding predictions and will guide nanobody selection. In this work, we demonstrate that INSPIRE-seq offers an unbiased approach to examine complex microenvironments and assist in the development of nanobodies, which could serve as active drugs, modified to become drugs, or used as targeting moieties.

bioengineering↗