Biology subjects

Rao, S. R.

Publications and source records attributed to Rao, S. R..

9 recordsLinked to original sources

bioRxiv2026

PACE, Proximity-Associated Changes in Expression

Cellular transcriptional states are shaped by local tissue context, yet quantifying how cellular gene expression varies with proximity to different cell types remains challenging. Cell-resolved spatial transcriptomics data are typically sparse and susceptible to contamination from neighbouring cells through diffusion, imperfect segmentation and cell overlap, making it difficult to distinguish genuine cell-state changes from technical artefacts. We present PACE (Proximity-Associated Changes in Expression), a hierarchical empirical Bayes framework for quantifying cell-type-resolved proximity effects on gene expression. PACE uses partial pooling to stabilise inference across genes and cell types, separates contamination from biologically meaningful spatial associations, and identifies coordinated transcriptional programs underlying each proximity effect. Applied to Xenium-profiled breast cancer tissue, PACE reveals tumour-associated reprogramming of stromal cells and macrophages at tumour interfaces. In CosMx-profiled melanoma, it identifies fibroblast responses to tumour proximity, including extracellular matrix programs that differ between tumours from patients with progressive and stable disease following immunotherapy. PACE provides a robust and interpretable framework for quantifying how tissue organisation shapes cellular state in spatial molecular data.

bioinformatics↗

bioRxiv2026

Assessing the effects of a 3D pathology tissue-processing workflow on downstream molecular analyses

Non-destructive 3D pathology methods have emerged in recent years with the potential to enhance standard 2D histopathology by greatly increasing the amount of tissue sampled by imaging and by providing volumetric morphological context. Another key advantage is that tissues remain intact, allowing re-embedding after imaging for potential long-term storage and future histological or molecular analyses. However, the impact of 3D pathology protocols on biomolecules -- including DNA, RNA, and proteins -- and their compatibility with downstream assays, has not been systematically evaluated. Here, we applied a previously optimized 3D pathology protocol -- involving deparaffinization, fluorescent H&E-analog staining, optical clearing, and open-top light-sheet microscopy -- to formalin-fixed paraffin-embedded (FFPE) specimens of breast, prostate, and head and neck cancer. Following the protocol, tissues were re-embedded in paraffin and compared with paired FFPE controls that did not undergo 3D pathology processing. DNA and RNA were extracted and subjected to quality assessments. Amplifiability was tested by PCR and reverse transcription quantitative PCR (RT-qPCR) of housekeeping genes. Although the results showed a slight decrease in the average yield and increased fragmentation of both DNA and RNA, amplifiability was largely preserved. Sanger sequencing of the PCR products confirmed accurate sequence determinations, while total RNA sequencing indicated that the global transcriptomic profile was largely unchanged. IHC staining of common biomarkers produced comparable signals, suggesting those proteins are well preserved after the 3D pathology workflow. These results demonstrate the feasibility of combining 3D pathology with downstream molecular applications.

pathology↗

bioRxiv2025

3D pathology-guided microdissection

Traditional micro- and macro-dissection techniques enable the extraction of localized regions in thin tissue sections for molecular analysis. Despite the growing use of 3D microscopy, analogous methods for volumetric microdissection are lacking. We have developed a 3D microdissection method based on computer numerical controlled (CNC) milling integrated with open-top light-sheet microscopy. We demonstrate the ability to study tumor evolution along convoluted 3D branching architectures, which is inaccessible to 2D methods.

bioengineering↗

bioRxiv2025

Bisphosphonates Trigger Anti-Ageing Effects Across Multiple Cell Types and Protect Against Senescence

Bisphosphonates (BPs) have been the major class of medicines used to treat disorders of excessive bone loss for over five decades. Recently it has been recognized that BPs may also have additional significant beneficial extra-skeletal effects. These include a reduction of all-cause mortality and of conditions commonly linked to ageing, such as cancer and cardiovascular disease. Here we show that bisphosphonates co-localize with lysosomal and endosomal organelles in non-skeletal cells and stimulate cell growth at low doses. In vivo spatial transcriptomic analysis revealed differentially expressed senescence markers in multiple organs of aged BP-treated mice, and a shift in cellular composition toward those of young counterparts. Similarly, a 5000-plex plasma proteome analysis from osteopenic patients before and after BP-treatment showed significant alterations in [~]400 proteins including GTPase regulators and markers of senescence, autophagy, apoptosis, and inflammatory responses. Furthermore, treatment with BPs protected against the onset of senescence in vitro. Proteome-wide target deconvolution using 2D thermal profiling revealed novel BP-binding targets (PHB2, ASAH1), and combined with RNA- and ATAC-seq of BP-treated cells and patient data, suggests downstream regulation of the MEF2A transcription factor within the heart. Collectively, these results indicate how BPs may beneficially modify the human plasma proteome, and directly impact multiple non-skeletal cell types through previously unidentified proteins, thereby influencing a range of pathways related to senescence and ageing.

cell biology↗

bioRxiv2025

Insights into global antimicrobial resistance dynamics through the sequencing of enteric bacteria from U.S. international travelers

Antimicrobial resistance (AMR) is an urgent threat to public health, but gaps in surveillance limit the detection of emergent novel threats and knowledge about the global distribution of AMR genes. International travelers frequently acquire AMR organisms, and thus may provide a window into AMR dynamics in otherwise poorly monitored regions and environments. To assess the utility of travelers as global AMR sentinels, we collected pre- and post-travel stool samples from 608 travelers, which were screened for the presence of extended-spectrum beta-lactamase producing Enterobacterales, carbapenem-resistant Enterobacterales, and mcr-mediated colistin-resistant Enterobacterales. A total of 307 distinct AMR organisms were sequenced in order to determine genotypic patterns and their association with travel region and behavior. Travel-associated AMR organisms were overwhelmingly E. coli, which exhibited considerable phylogenetic diversity regardless of travel region. However, the prevalence of resistance genes varied by region, with blaCTX-M-55 and blaCTX-M-27 significantly more common in travelers returning from South America and South-Eastern Asia, respectively. Hybrid assembly and plasmid reconstruction revealed the genomic neighborhood of blaCTX-M-55 frequently matched a motif previously linked to animal populations. Contact with animals was also associated with virulence factors in acquired AMR organisms, including carriage of the ColV plasmid, a driver of avian pathogenic E. coli. We identified novel variants of the mcr-1 gene in strains acquired from Western Africa, highlighting the potential for traveler surveillance to detect emerging clinical threats. Ongoing efforts to track travel-acquired organisms could complement existing global AMR surveillance frameworks. ImportanceWe collected pre- and post-travel stool samples from 608 U.S. international travelers, which were screened for the presence of three clinically relevant antimicrobial-resistant organisms, with the goal of understanding the utility of travelers as sentinels for emerging AMR and identifying regional resistance patterns. We sequenced 307 organisms and uncovered significant diversity and geographic heterogeneity in AMR organisms. We found significant differences in certain resistance gene alleles of travelers returning from South America and South-Eastern Asia, traveler contact with animals associated with virulence factors, including a plasmid that is a known driver of avian pathogenic E. coli, and novel variants of the colistin resistance mcr-1 gene in strains acquired from Western Africa. This work emphasizes the value of monitoring international travelers as a proxy for understanding AMR patterns globally and could be an important addition to existing global AMR surveillance frameworks.

genomics↗

bioRxiv2025

High cholesterol increases myeloma tumour burden and promotes resistance to bortezomib

Multiple myeloma (MM) is an incurable hematologic cancer where malignant plasma cells accumulate in the bone marrow (BM), with disease progression highly dependent on cellular interactions. Obesity is a major risk factor for myeloma, however the underlying mechanisms are unclear. Here we used a murine myeloma model to show that a high cholesterol diet increases local and circulating levels of low density lipoprotein cholesterol (LDL), increasing bone marrow tumour burden in vivo. Exogenous LDL induced bortezomib-specific drug resistance in metabolically stressed myeloma cells in vitro and ex vivo. RNA-seq analysis revealed bortezomib-induced changes in the cholesterol biosynthesis/homeostasis pathway that were completely reverted with LDL pretreatment. In silico analysis of patient data supported a role for cholesterol in response to bortezomib-based therapies. Targeted proteome profiling revealed changes in expression of the adipokine resistin in the bone marrow of cholesterol-treated or myeloma-bearing mice, with elevated resistin expression observed in MGUS patients. Bone marrow adipocytes were found to be a major source of resistin, which was further increased in response to LDL. In summary, we demonstrate that high cholesterol promotes both myeloma development and bortezomib resistance, identifying resistin as a potential mediator so revealing new mechanisms underlying myeloma pathogenesis.

cancer biology↗

bioRxiv2023

Molecular analysis of archival diagnostic prostate cancer biopsies identifies genomic similarities in cases with progression post-radiotherapy, and those with de novo metastatic disease.

PurposeIt is important to identify molecular features that improve prostate cancer (PCa) risk stratification before radical treatment with curative intent. Molecular analysis of historical diagnostic formalin-fixed paraffin-embedded (FFPE) prostate biopsies from cohorts with post-radiotherapy (RT) long-term clinical follow-up has been limited. Utilizing parallel sequencing modalities, we performed a proof-of-principle sequencing analysis of historical diagnostic FFPE prostate biopsies. We compared patients with i) stable PCa post-primary or salvage RT (sPCa), ii) progressing PCa post-RT (pPCa), and iii) de novo metastatic PCa (mPCa). Experimental DesignA cohort of 19 patients with diagnostic prostate biopsies (n=6 sPCa, n=5 pPCa, n=8 mPCa) and mean 4 years 10 months follow-up (diagnosed 2009-2016) underwent nucleic acid extraction from demarcated malignancy. Samples underwent 3RNA sequencing (3RNAseq) (n=19), nanoString analysis (n=12) and Illumina 850k methylation (n=8) sequencing. Bioinformatic analysis was performed to coherently identify differentially expressed genes (DEGs) and methylated genomic regions (MGRs). Results18 of 19 samples provided useable 3RNAseq data. Principal Component Analysis (PCA) demonstrated similar expression profiles between pPCa and mPCa cases, versus sPCa. Coherently differentially methylated probes between these groups identified [~]600 differentially MGRs. The top 50 genes with increased expression in pPCa patients were associated with reduced progression-free survival post-RT (p<0.0001) in an external cohort. Conclusions3RNAseq, nanoString and 850K-methylation analyses are each achievable from historical FFPE diagnostic pre-treatment prostate biopsies, unlocking the potential to utilize large cohorts of historic clinical samples. Profiling similarities between individuals with pPCa and mPCa suggests biological similarities and historical radiological staging limitations, which warrant further investigation.

genomics↗

bioRxiv2023

Intra-prostatic tumour evolution, steps in metastatic spread and histogenomic associations revealed by integration of multi-region whole genome sequencing with histopathological features

Extension of prostate cancer beyond the primary site into the surrounding organs by local invasion or nodal metastasis is associated with poor prognosis. The emergence and evolution of cancer clones at this early stage of expansion and spread has not been studied in detail. We performed whole genome sequencing on 42 prostate cancer samples from the prostate, seminal vesicles and regional lymph nodes of five treatment-naive patients with locally advanced disease who underwent radical prostatectomy. Using cancer cell fractions computed from single nucleotide variants and copy number alterations, we reconstructed the tumour phylogenies, which in turn allowed us to infer key molecular steps in the progression of prostate cancer in these individuals. We mapped the clonal composition of cancer sampled across the prostate in each individual and inferred the routes of spread of cancer cells within the prostate and to seminal vesicles and lymph nodes. Based on these data, we delineated the route of tumour progression and metastasis following the transformation of adenocarcinoma to amphicrine morphology, the molecular events leading to whole genome duplication associated with a single clonal expansion and identified putative driver events associated with local invasion and lymph node metastasis. We also correlated genomic changes associated with differences in morphology and identified putative driver events associated with spread to seminal vesicle invasion and lymph node metastasis. Taken together, these findings have implications for diagnosis and risk stratification, in addition to providing a rationale for further studies to characterise the genetic changes associated with morphological transformation. Our results demonstrate the value of integrating multi-region sequencing with histopathological data to study tumour evolution and identify mechanisms of prostate cancer spread.

cancer biology↗

bioRxiv2020

A cross-species drug discovery pipeline to identify and validate new treatments for osteosarcoma

PurposeOsteosarcoma is a rare but aggressive bone cancer that occurs primarily in children. Like other rare cancers, treatment advances for osteosarcoma have stagnated, with little improvement in survival for the past several decades. Developing new treatments has been hampered by extensive genomic heterogeneity and limited access to patient samples to study the biology of this complex disease. Experimental designTo overcome these barriers, we combined the power of comparative oncology with patient-derived models of cancer and high-throughput chemical screens in a cross-species drug discovery pipeline. ResultsCoupling in vitro high-throughput drug screens on low-passage and established cell lines with in vivo validation in patient-derived xenografts we identify the proteasome and CRM1 nuclear export pathways as therapeutic sensitivities in osteosarcoma, with dual inhibition of these pathways inducing synergistic cytotoxicity. ConclusionsThese collective efforts provide an experimental framework and set of new tools for osteosarcoma and other rare cancers to identify and study new therapeutic vulnerabilities.

cancer biology↗