Biology subjects

Brown, D. D.

Publications and source records attributed to Brown, D. D..

12 recordsLinked to original sources

bioRxiv2026

Immunotherapy restores therapeutic efficacy of fecal microbiome transplants to treat Clostridioides difficile

Microbiome-based therapeutics to treat enteric bacterial infections requires stable engraftment of transplanted beneficial bacteria that target and eliminate the pathogen. The host factors that determine whether a microbiome transplant engrafts remain largely unresolved. Here we demonstrate that Interleukin-10 (IL-10) signaling deficiency leads to impaired fecal microbiota transplantation (FMT) engraftment and failure to resolve Clostridioides difficile infection in mice. In the absence of IL-10-mediated immunoregulation provided by Foxp3+ Treg cells, increased IFN-{gamma} signaling in the intestine leads to elevated production of reactive oxygen/nitrogen species (ROS/RNS) by neutrophils and epithelial cells that supports the growth of inflammation-tolerant microbes, inhibits FMT engraftment, and impairs resolution of C. difficile. FMT treatment success can be restored by antibody-mediated blockade of IFN-{gamma} signaling, neutrophil depletion or inhibition of ROS/RNS production. Lastly, we developed an Il10-mRNA-LNP immunotherapy to boost IL-10 signaling in FMT Non-Responsive mice and demonstrate that Il10-mRNA-LNP administration is sufficient to restore FMT engraftment and subsequent resolution of C. difficile. Combined, these data support a mechanism by which IL-10 released by Treg cells limits IFN-{gamma} mediated intestinal inflammation thereby promoting an intestinal microenvironment receptive to FMT engraftment and resolution of C. difficile. These data demonstrate that the host's immune status can be therapeutically modulated to improve microbiome-based approaches to treat infection.

immunology↗

bioRxiv2026

Benchmarking scRNA-seq Copy Number Inference: A Comprehensive Evaluation and Practitioner Guide

Accurately inferring copy number variation (CNV) from scRNA-seq data is critical for identifying malignant cells, reconstructing tumor subclonal architecture, and uncovering the genomic drivers that dictate cancer cell biology. However, the performance of existing tools varies significantly, and current benchmarks lack the breadth of datasets and methods necessary to provide definitive guidance. We present a comprehensive benchmark of 12 CNV inference methods across 28 real datasets (>100,000 cells) and diverse synthetic datasets. By evaluating methods based on malignant cell classification accuracy, CNV inference accuracy, scalability, and robustness, we establish a definitive practitioners guideline: allele-aware methods like Numbat excel when high-quality allelic inference can be achieved, whereas expression-centric tools such as Clonalscope, CopyKAT, inferCNV, and SCEVAN remain reliable when raw sequencing data are unavailable. Our study provides both a practical decision-making framework for researchers and a public repository of standardized CNV profiles to catalyze further methodological innovation.

cancer biology↗

bioRxiv2026

Distinct p21 dynamics drive alternative routes to whole-genome duplication through a common CDK4/6-dependent polyploid G0 state

Whole-genome duplication (WGD) fuels tumor evolution and therapy resistance, yet the molecular mechanisms governing the switch from the canonical mitotic cell cycle to the endoreplication cycle remain unclear. Here, we combine single-cell proteomics, manifold learning, and live-cell imaging to map the intersection of the mitotic and endoreplication cycles in breast cancer cells exposed to genotoxic agents. We identify two distinct routes to WGD driven by distinct p21 dynamics. High p21 induction induces G2 exit and endocycling, whereas insufficient p21 permits mitotic entry followed by slippage and endomitosis. This therapy-induced switch acts as a facultative stress response, generating drug-resistant polyploid populations that propagate genomic instability through replication stress and the generation of replication-competent micronuclei. Both paths to WGD converge on a common polyploid G0 state dependent on cyclin D1:CDK4/6 activity to complete the transition to the endoreplication cycle, revealing a shared vulnerability. Sequential treatment with genotoxic agents followed by CDK4/6 inhibitors preserves the cytotoxic efficacy of DNA-damaging drugs while simultaneously blocking entry into the endoreplication cycle and WGD-driven evolutionary rescue. These findings reveal the molecular rules governing the switch from the mitotic to endoreplication cycle and highlight the potential of WGD-blocking drugs as adjuvant therapies to inhibit drug resistance and suppress tumor evolution.

cell biology↗

bioRxiv2025

Identification and Credentialing of Patient Derived Xenograft Models of Invasive Lobular Breast Carcinoma using Multi-omics and Histopathology assessment

Breast cancer is a heterogeneous disease with numerous histological subtypes. Invasive lobular cancer (ILC) is the most common special subtype, accounting for 10-15% of all breast cancers. The pathognomonic feature of ILC is the loss of E-cadherin (CDH1), which leads to a unique single-file growth pattern of discohesive cells. Although ILCs show better prognostic factors than the most common No Special Type (NST) of breast cancer, patients with ILC have worse long-term outcomes, which is not well understood. In this study, we aimed to identify and characterize Patient-Derived Xenograft (PDX) models of ILC based upon the presence of truncating CDH1 mutations and/or low CDH1 mRNA expression among 128 human breast cancer PDX models. We selected 8 PDX models for validation using Immunohistochemical (IHC) analysis for E-Cadherin, p120, ER, PR, and HER2. We confirmed that seven of these PDX models are indeed ILC while one was identified as mixed NST-ILC PDX. Molecular analysis of the confirmed ILC PDX models showed enrichment of truncating CDH1 mutations, significantly lower levels of CDH1 mRNA expression and predominantly luminal subtypes compared to NST PDX models, in line with the molecular characteristics of human ILC disease. The commonly altered genes in the ILC PDX models included PIK3CA (57%), CDH1 (57%) and TP53 (57%) among others. Our study confirms and characterizes new ILC PDX models, offering valuable tools to advance our understanding of human ILC biology and support the development of innovative treatment strategies.

cancer biology↗

bioRxiv2025

Integrating Artificial Intelligence-Driven Digital Pathology and Genomics to Establish Patient-Derived Organoids as a Novel Alternative Model for Drug Response in Head and Neck Cancer

Patient-derived organoids (PDOs) are emerging as advanced 3D ex vivo novel alternative method (NAM) preclinical models, offering significant advantages over traditional cell lines and monolayer cultures for therapeutic development. In this study, we established PDOs from surgically resected fresh tissues of human papillomavirus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) across anatomical sites, tumor T-categories, and sample types. These PDOs faithfully recapitulate the tumors pathology, mutational profile, and drug response. To enable rapid classification of PDO identity, we developed a new convolutional neural network (CNN) model, TransferNet-PDO, which accurately distinguished tumor versus normal PDOs in culture using digital histopathology images (AUC[≥]0.88). PDOs maintained stable cultures and were cryopreserved between passages 5 and 12. Immunohistochemistry (IHC) staining (PanCK, p63, Cytokeratin 13, Ki67) confirmed squamous phenotype and histologic aggression of the original tumor. For tumors harboring TP53 mutations by whole-exome sequencing (WES), PDOs retained the corresponding p53 functional status as confirmed by IHC (enhanced or loss of expression). Somatic mutational landscape revealed that PDOs preserved driver somatic mutations, copy number variations (CNVs), and clonal architecture including low-prevalence subclones. Drug sensitivity assessment of PDOs showed that cisplatin reduced cell viability, whereas cetuximab and lenvatinib had minimal effects. Chemoradiation led to greater tumor organoid killing compared to radiation or chemotherapy alone. This study presents an integrated HNSCC PDO platform combining tissue biobanking, organoid establishment, multi-omics characterization, functional drug screening, and AI-driven histopathologic classification, providing a comprehensive and scalable system for translational cancer research.

cancer biology↗

bioRxiv2025

Role of the NuRD complex and altered proteostasis in cancer cell quiescence

Cytotoxic chemotherapy remains the primary treatment for ovarian cancer (OvCa). Development of chemoresistance typically results in patient death within two years. As such, understanding chemoresistance is critical. One underexplored mechanism of chemotherapy resistance is quiescence. Quiescent cells, which have reversibly exited the cell cycle, are refractory to most chemotherapies which primarily target rapidly proliferating cells. Here, we report that CHD4 and MBD3, components of the nucleosome remodeling and deacetylase (NuRD) complex, are downregulated in quiescent OvCa cells (qOvCa). Indicating a direct role for NuRD complex downregulation in the induction of quiescence, either CHD4 or MBD3 knockdown or histone deacetylase inhibitors (HDACi), such as vorinostat, induce quiescence in OvCa cells. RNA-Seq analysis of HDACi-treated cells confirmed expression changes consistent with induction of quiescence. We also find that both primary qOvCa and vorinostat-induced qOvCa demonstrate altered proteostasis, including increased proteasome activity and autophagy, and combination therapy of HDACi and proteasome inhibitors or autophagy inhibitors demonstrated profound synergistic death of OvCa cells. Finally, we overlapped RNA-Seq signatures from quiescent ovarian cancer cells with genes essential for quiescence in yeast to identify a "quiescent cell core signature." This core quiescent cell signature appeared to be conserved across multiple cancer types, suggesting new therapeutic targets.

cancer biology↗

bioRxiv2024

Identification of the MRTFA/SRF pathway as a critical regulator of quiescence in cancer.

Chemoresistance is a major driver of cancer deaths. One understudied mechanism of chemoresistance is quiescence. We used single cell culture to identify, retrieve, and RNA-Seq profile primary quiescent ovarian cancer cells (qOvCa). We found that many qOvCa differentially expressed genes are transcriptional targets of the Myocardin Related Transcription Factor/Serum Response Factor (MRTF/SRF) pathway. We also found that genetic disruption of MRTF-SRF interaction, or an MRTF/SRF inhibitor (CCG257081) impact qOvCa gene expression and induce a quiescent state in cancer cells. Suggesting a broad role for this pathway in quiescence, CCG257081 treatment induced quiescence in breast, lung, colon, pancreatic and ovarian cancer cells. Furthermore, CCG081 (i) maintained a quiescent state in patient derived breast cancer organoids and, (ii) induced tumor growth arrest in ovarian cancer xenografts. Together, these data suggest that MRTF/SRF pathway is a critical regulator of quiescence in cancer and a possible therapeutic target. SignificanceQuiescence is a critical driver of chemoresistance. The MRFT-SRF pathway regulates cancer cell quiescence and inhibiting the MRTF-SRF pathway can prevent the outgrowth of quiescent cancer cells and improve cancer outcomes.

cancer biology↗

bioRxiv2024

Hope for Others: Research Results from the University of Pittsburgh Rapid Autopsy Program for Breast Cancer

Breast cancer affects 1/8 of women throughout their lifetimes, with over 90% of cancer deaths being caused by metastasis. However, metastasis poses unique challenges to research, as complex changes in the microenvironment in different metastatic sites and difficulty obtaining tissue for study hinder the ability to examine in depth the changes that occur during metastasis. Rapid autopsy programs thus fill a unique need in advancing metastasis research. Here, we describe our protocol and processes for establishing and improving the US-based Hope for OTHERS (Our Tissue Helping Enhance Research and Science) program for organ donation in metastatic breast cancer. As of August 2024, we consented 114 patients and performed 37 autopsies, from which we collected 551 unique metastatic frozen tumor samples, 1244 FFPE blocks, 90 longitudinal liquid biopsy samples and developed 14 patient-derived organoid and 8 patient-derived xenograft models. We report in-depth clinical and histopathological information and discuss extensive new research and novel findings in patient outcomes, metastatic phylogeny, and factors in successful living model development. Our results reveal key logistical and protocol improvements that are uniquely beneficial to certain programs based on identifiable features, such as working closely with patient advocates, methods to rescue RNA quality in cases where tissue quality may degrade due to time delays, as well as guidelines and future expansions of our program. Statement of SignificanceRapid autopsy programs are unique research settings with huge potential for studying metastatic cancer, however, they have complex research challenges. Our work provides a valuable resource in advancing this field of research.

cancer biology↗

bioRxiv2024

Systemic and local chronic inflammation and hormone disposition promote a tumor-permissive environment for breast cancer in older women

Estrogen receptor positive (ER+) breast cancer is the most common subtype of breast cancer and is an age-related disease. The peak incidence of diagnosis occurs around age 70, even though these post-menopausal patients have low circulating levels of estradiol (E2). Despite the hormone sensitivity of age-related tumors, we have a limited understanding of the interplay between systemic and local hormones, chronic inflammation, and immune changes that contribute to the growth and development of these tumors. Here, we show that aged F344 rats treated with the dimethylbenz(a)anthracene / medroxyprogestrone acetate (DMBA/MPA) carcinogen develop more tumors at faster rates than their younger counterparts, suggesting that the aged environment promotes tumor initiation and impacts growth. Single-nuclei RNA-seq (snRNA-seq) of the tumors showed broad local immune dysfunction that was associated with circulating chronic inflammation. Across a broad cohort of specimens from patients with ER+ breast cancer and age-matched donors of normal breast tissue, we observe that even with an estrone (E1)-predominant estrogen disposition in the systemic circulation, tumors in older patients increase HSD17B7 expression to convert E1 to E2 in the tumor microenvironment (TME) and have local E2 levels similar to pre-menopausal patients. Concurrently, trackable increases in several chemokines, defined most notably by CCL2, promote a chronically inflamed but immune dysfunctional TME. This unique milieu in the aged TME, characterized by high local E2 and chemokine-enriched chronic inflammation, promotes both accumulation of tumor-associated macrophages (TAMs), which serve as signaling hubs, as well as polarization of TAMs towards a CD206+/PD-L1+, immunosuppressive phenotype. Pharmacologic targeting of estrogen signaling (either by HSD17B7 inhibition or with fulvestrant) and chemokine inflammation both decrease local E2 and prevent macrophage polarization. Overall, these findings suggest that chronic inflammation and hormonal disposition are critical contributors to the age-related nature of ER+ breast cancer development and growth and offer potential therapeutic insight to treat these patients. Translational SummaryWe uncover the unique underpinnings establishing how the systemic host environment contributes to the aged breast tumor microenvironment, characterized by high local estradiol and chronic inflammation with immune dysregulation, and show that targeting avenues of estrogen conversion and chronic inflammation work to restore anti-tumor immunity. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/616978v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@1ecd90borg.highwire.dtl.DTLVardef@9d7ac8org.highwire.dtl.DTLVardef@e7fb74org.highwire.dtl.DTLVardef@2da524_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2023

Small molecule nitroalkenes inhibit RAD51-mediated homologous recombination and amplify triple-negative breast cancer cell killing by DNA-directed therapies.

Nitro fatty acids (NO2-FAs) are endogenously generated lipid signaling mediators from metabolic and inflammatory reactions between conjugated diene fatty acids and nitric oxide or nitrite-derived reactive species. NO2-FAs undergo reversible Michael addition with hyperreactive protein cysteine thiolates to induce posttranslational protein modifications that can impact protein function. Herein, we report a novel mechanism of action of natural and non-natural nitroalkenes structurally similar to (E) 10-nitro-octadec-9-enoic acid (CP-6), recently de-risked by preclinical Investigational New Drug-enabling studies and Phase 1 and Phase 2 clinical trials and found to induce DNA damage in a TNBC xenograft by inhibiting homologous-recombination (HR)-mediated repair of DNA double-strand breaks (DSB). CP-6 specifically targets Cys319, essential in RAD51-controlled HR-mediated DNA DSB repair in cells. A nitroalkene library screen identified two structurally different nitroalkenes, a non-natural fatty acid [(E) 8-nitro- nonadec-7-enoic acid (CP-8)] and a dicarboxylate ester [dimethyl (E)nitro-oct-4-enedioate (CP- 23)] superior to CP-6 in TNBC cells killing, synergism with three different inhibitors of the poly ADP-ribose polymerase (PARP) and {gamma}-IR. CP-8 and CP-23 effectively inhibited {gamma}-IR-induced RAD51 foci formation and HR in a GFP-reported assay but did not affect benign human epithelial cells or cell cycle phases. In vivo, CP-8 and CP-23s efficacies diverged as only CP-8 showed promising anticancer activities alone and combined with the PARP inhibitor talazoparib in an HR-proficient TNBC mouse model. As preliminary preclinical toxicology analysis also suggests CP-8 as safe, our data endorse CP-8 as a novel anticancer molecule for treating cancers sensitive to homologous recombination-mediated DNA repair inhibitors. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=172 SRC="FIGDIR/small/552990v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@1dcec56org.highwire.dtl.DTLVardef@1202a09org.highwire.dtl.DTLVardef@a7e7b6org.highwire.dtl.DTLVardef@15d8925_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2023

EstroGene database reveals diverse temporal, context-dependent and directional estrogen receptor regulomes in breast cancer

As one of the most successful cancer therapeutic targets, estrogen receptor- (ER/ESR1) has been extensively studied in decade-long. Sequencing technological advances have enabled genome-wide analysis of ER action. However, reproducibility is limited by different experimental design. Here, we established the EstroGene database through centralizing 246 experiments from 136 transcriptomic, cistromic and epigenetic datasets focusing on estradiol-treated ER activation across 19 breast cancer cell lines. We generated a user-friendly browser (https://estrogene.org/) for data visualization and gene inquiry under user-defined experimental conditions and statistical thresholds. Notably, documentation-based meta-analysis revealed a considerable lack of experimental details. Comparison of independent RNA-seq or ER ChIP-seq data with the same design showed large variability and only strong effects could be consistently detected. We defined temporal estrogen response metasignatures and showed the association with specific transcriptional factors, chromatin accessibility and ER heterogeneity. Unexpectedly, harmonizing 146 transcriptomic analyses uncovered a subset of E2-bidirectionally regulated genes, which linked to immune surveillance in the clinical setting. Furthermore, we defined context dependent E2 response programs in MCF7 and T47D cell lines, the two most frequently used models in the field. Collectively, the EstroGene database provides an informative resource to the cancer research community and reveals a diverse mode of ER signaling.

cancer biology↗

bioRxiv2022

Loss of E-cadherin Induces IGF1R Activation Revealing a Targetable Pathway in Invasive Lobular Breast Carcinoma

No Special Type breast cancer (NST; commonly known as Invasive Ductal Carcinoma (IDC)) and Invasive Lobular Carcinoma (ILC) are the two major histological subtypes of breast cancer with significant differences in clinicopathological and molecular characteristics. The defining pathognomonic feature of ILC is loss of cellular adhesion protein, E-cadherin (CDH1). We have previously shown that E-cadherin functions as a negative regulator of the Insulin-like Growth Factor 1 Receptor (IGF1R) and propose that E-cadherin loss in ILC sensitizes cells to growth factor signaling which thus alters their sensitivity to growth factor signaling inhibitors and their downstream activators. To investigate this potential therapeutic vulnerability, we generated CRISPR-mediated CDH1 knockout (CDH1 KO) IDC cell lines (MCF7, T47D, ZR75.1) to uncover the mechanism by which loss of E-cadherin results in IGF pathway activation. CDH1 KO cells demonstrated enhanced invasion and migration that was further elevated in response to IGF1, serum and Collagen I. CDH1 KO cells exhibited increased sensitivity to IGF resulting in elevated downstream signaling. Despite minimal differences in membranous IGF1R levels between wildtype (WT) and CDH1 KO cells, significantly higher ligand-receptor interaction was observed in the CDH1 KO cells, potentially conferring enhanced downstream signaling activation. Critically, increased sensitivity to IGF1R, PI3K, Akt and MEK inhibitors was observed in CDH1 KO cells and ILC patient-derived organoids, suggesting that these targets require further exploration in ILC treatment and that CDH1 loss may be exploited as a biomarker of response for patient stratification.

cancer biology↗