Biology subjects

Clarke, R. B.

Publications and source records attributed to Clarke, R. B..

11 recordsLinked to original sources

bioRxiv2026

An in vitro model of breast cancer metastatic niche priming

Metastatic breast cancer is responsible for around 11,500 deaths a year in the UK. The primary tumour likely plays a major role in priming the distant site for metastasis and crosstalk between primary and metastatic sites may be essential for secondary tumour growth. We have developed a novel in vitro model in which we can further study these interactions; evaluating niche priming and cancer cell conditioning as well as assessing their influence on cell homing and colonisation. In this paper we describe a model that we believe adds to the array of in vitro tools available to study various stages of the metastatic cascade, offering a unique opportunity to assess bidirectional, primary to niche interactions in vitro. We show that proliferation, migration and chemotaxis, and stem cell activity are altered in both cancer cell lines and in lung epithelial cells following linked, fluidic culture. Changes in cell homing and colonisation can be modelled in cell lines and within viable lung tissue explants taken from mice, with breast cancer cells settling and growing within the lung epithelial cells and tissue explants over 7 days. The colonisation/growth of cells injected into the system closely represents that seen following tail vein injection and cancer cells can be seen to settle and grow within the lung epithelial cells.

cancer biology↗

bioRxiv2026

Selection for targeted therapy resistance leads to an indirect selection for higher phenotypic plasticity and enhanced evolvability to orthogonal stressors

Acquired resistance to targeted therapies is a primary barrier to durable cancer control. Resistance frequently coincides with stem-like features and EMT-associated cell-state changes (partial EMT), yet whether these programs directly cause resistance or instead enable escape by increasing phenotypic plasticity remains debated. Integrating computational modeling, functional experimental assays, and lineage tracing, we investigated how cell-state plasticity shapes acquired resistance to ALK inhibition in ALK+ lung cancer models. Our results support a model in which drug exposure selects for cells with higher phenotypic plasticity, progressively increasing their representation as resistance emerges. Consequently, resistant populations show enhanced capacity to adapt to orthogonal therapeutic and environmental stressors and exhibit heightened metastatic potential. Bulk ATAC-seq showed that highly plastic cells have increased chromatin accessibility at regulators of EMT and stemness. Consistent with this, boosting plasticity via Yamanaka-factor induction or EMT-factor expression reduced ALKi sensitivity over time. In contrast, constraining plasticity (SOX2 knockdown or epigenetic inhibition) reduced long-term resistance outgrowth and prolonged ALKi response. Together, our results indicate that targeted therapy indirectly selects for cells with increased phenotypic plasticity, providing the substrate from which multifactorial resistance and metastatic competence evolve. Further, it suggests that constraining plasticity could delay resistance and extend response durability.

cancer biology↗

bioRxiv2026

Grading HER2 at the nanoscale in clinical tissue

To guide diagnosis and treatment, breast cancer biopsies are assessed for HER2 status and assigned one of four grades (0-3+). While current practices are sufficient for detection of HER2 overexpression (3+), there is a need for more sensitive methods capable of characterising lower HER2 expression in patients who may still benefit from HER2-targeted therapies. Super-resolution fluorescence microscopy techniques, such as single molecule localisation microscopy (SMLM), have reshaped the study of nanoscale molecular architecture by visualising single target molecules in a range of sample types. Here, we have developed a quantitative SMLM workflow to visualise HER2 nanoclustering in patient-derived xenografts (PDX) and clinical breast tumour tissue from eight patients spanning all disease grades. Analysis of HER2 cluster architecture revealed grade-dependent changes in size of cluster and HER2 abundance. We then applied a blinded data-driven approach to regroup samples based on this nanoscale HER2 clustering. This led to the reclassification of three samples into new groups, due to similarities in nanoscale signature. Together, these findings demonstrate that quantitative fluorescence nanoscopy can be used to identify clinical HER2 phenotypes across a range of expression levels due to its exquisite sensitivity, and this could be leveraged to stratify patients for targeted therapy.

cancer biology↗

bioRxiv2025

Label-free DIA-NN enabled RIME (DIANNeR) reveals glucocorticoid receptor interaction networks in breast, bladder, and blood across normaluntransformed cells, cancer cell lines, and PDXs

Understanding how transcription factors execute tissue-specific programmes requires defining their protein interaction networks in physiologically relevant contexts. However, profiling normal untransformed cells presents fundamental challenges due to intrinsically limited input material. To meet this challenge we integrated data-independent acquisition (DIA), ion mobility separation, and library-free analysis to achieve a 2-fold increase in detection without loss in enrichment. Applied to the glucocorticoid receptor (GR), a ubiquitously expressed nuclear receptor driving pleiotropic responses to standard-of-care anti-inflammatory therapeutics, DIANNeR (DIA-NN enabled RIME) resolves distinct, context-dependent networks across breast, ureter and blood in normal and transformed contexts. Key findings include: detection of a HOXA5-GR interaction in normal epithelium which was undetected in malignant models; an epithelial-restricted SMARCD3-GR interaction with prognostic relevance; and a FOXP3-BCL11B-GR network in primary CD4+ T cell populations undetected by data dependant acquisition (DDA). By defining normal GR tissue interactomes, DIANNeR provides the essential comparator for interpreting network rewiring that drives disease.

cancer biology↗

bioRxiv2025

HES1 oscillations are required for cell cycle progression and re-entry in oestrogen receptor positive breast cancer cells

Long-term recurrence in breast cancer is driven by reactivation of dormant disseminated tumour cells (DTCs) and remains a major clinical challenge, particularly in oestrogen receptor positive (ER) tumours. This process is underpinned by regulation of the cell cycle machinery that controls quiescence maintenance and exit. HES1, a Notch pathway transcription factor, regulates key cell cycle genes and has been shown to demonstrate protein expression oscillations. Here, we sought to establish whether HES1 oscillations may regulate ER+ cancer cell quiescence and reactivation. To investigate this, we developed a fundamental in vitro model of cell cycle arrest and re-entry based on reversible CDK4/6 inhibition (CDK4/6i) with palbociclib, compatible with quantitative single-cell live-imaging of a knock-in endogenous HES1 reporter. Consistent with earlier findings, HES1 exhibited [~]24-hour protein oscillations in cycling cells demonstrating a reproducible dip in protein expression prior to S-Phase. During CDK4/6i-mediated arrest, the [~]24h HES1 oscillation was lost, HES1 levels were maintained at a moderately higher level and HES1 exhibited smaller dips. Similar changes were observed in unperturbed, spontaneously quiescent cells. Following release from CDK4/6i and cell cycle re- entry, these alterations were reversed and the characteristic G1/S HES1 dip was observed. Preventing this dip at the point of release, by inducibly sustaining HES1 with a Tet-On system, upregulated the cell cycle inhibitor p21, impeded cell cycle re-entry and induced cell death. These findings suggest that manipulating HES1 dynamics could represent a promising therapeutic approach to prevent reactivation of dormant tumour cells. Significance StatementBreast cancer can recur years after initial treatment due to reactivation of dormant tumour cells. Understanding how these cells exit dormancy is crucial for preventing relapse. We investigated HES1, a transcription factor with rhythmic protein oscillations, and its role in regulating quiescence in oestrogen receptor-positive (ER) breast cancer cells. Using live-cell imaging and a reversible cell cycle arrest model, we show that HES1 dynamics change during dormancy and reactivation, and that disrupting these oscillations prevents cell cycle re-entry and induces cell death. These findings reveal HES1 protein dynamics as a potential therapeutic vulnerability and highlight a novel strategy to target dormant cancer cells to prevent their reactivation.

cancer biology↗

bioRxiv2025

Progesterone Receptor Expressing Basal Cells and Progesterone Receptor-Primed Progenitors Redefine the Mammary Epithelial Hierarchy

The central role of progesterone in breast biology and cancer is undeniable. Progesterone is a potent mitogen for mammary stem cell expansion and essential for mammopoiesis, yet only a fraction of the luminal epithelium is described to express the progesterone receptor (PR). Progestins in contraceptives and HRT regimens increases breast cancer risk, whilst progesterone inhibition reduces mammary tumorigenesis. Understanding PR within mammary epithelial dynamics is imperative, especially given mammary stem/progenitors cells are the cells-of-origin in breast cancer. Here, we show PR-primed progenitors contribute to both mammary lineages and expose a novel PR+ basal population, challenging current dogma in the field. PR lineage-tracing yields an unprecedented contribution to the luminal and basal compartments. We uncover an asymmetrically dividing PR-primed subpopulation, which has unique bipotent clonogenic capacity and forms TEB-like outgrowths upon transplantation. We enumerate PR+ basal cells and dissect their proteomic landscape, establishing PR+ basal cells as a discrete basal subset, disparate from luminal PR+ cells. Finally, we identify PR+ basal cells in multiple proteomes, scRNAseq datasets and localize PR in clinical breast specimens. This forms a new foundation for comprehending hormone receptor patterning in the breast, not only based on lineage-identity but also the progenitor-progeny hierarchy. Our study shifts the current paradigm of breast biology with implications for breast cancer treatment given the growing interest in anti-progestin based primary prevention strategies.

cell biology↗

bioRxiv2025

MYBL2 maintains stemness and promotes theta-mediated end joining in triple negative breast cancer

Triple negative breast cancer (TNBC) has a poor prognosis due to limited treatment options and high metastasis risk. Breast cancer (BC) stem/progenitor cells, also known as tumour initiating cells, are a small, difficult to target population within the tumour responsible for metastasis and therapy resistance. MYBL2 overexpression is commonly linked to poor prognosis and metastasis but its role in BC stem/progenitor cells remains elusive. To determine the role of MYBL2 in BC stem/progenitor cells we generated TNBC cell lines with inducible MYBL2 downregulation. Our findings reveal that elevated MYBL2 is essential for self-renewal, DNA repair, and replication stress response in these cells and lowering MYBL2 impairs stemness and self-renewal both in vitro and in vivo. Accordingly, our functional and mechanistic analyses indicate that high-MYBL2 stem/progenitor cells exhibit increased sensitivity to Pol{theta} inhibitors which is lost upon MYBL2 downregulation due to transcriptional suppression. Combining Pol{theta} inhibitors with ATR inhibitors further enhances this sensitivity, in vitro and ex vivo. This study thus identifies Pol{theta}/ATR inhibition as a synthetic lethality strategy to eradicate BC stem/progenitor cells and underscores MYBL2 expression as a biomarker for patient stratification in this treatment.

cancer biology↗

bioRxiv2021

Reciprocal Priming between RTKs within Recycling Endosomes Orchestrates Cellular Signaling Outputs

Integration of signalling downstream of individual receptor tyrosine kinases (RTKs) is crucial to fine tune cellular homeostasis during development and in pathological conditions, including breast cancer. However, how signalling integration is regulated and whether the endocytic fate of single receptors controls such signalling integration remains poorly elucidated. Combining quantitative phosphoproteomics and targeted assays, we generated a detailed picture of recycling-dependent fibroblast growth factor (FGF) signalling in breast cancer cells, with a focus on distinct FGF receptors (FGFRs). We discovered reciprocal priming between FGFRs and epidermal growth factor (EGF) receptor (EGFR) that is coordinated at recycling endosomes. FGFR recycling ligands induce EGFR phosphorylation on threonine 693. This phosphorylation event alters both FGFR and EGFR trafficking and primes FGFR-mediated proliferation but not cell invasion. In turn, FGFR signalling primes EGF-mediated outputs via EGFR threonine 693 phosphorylation. This reciprocal priming between distinct families of RTKs from recycling endosomes exemplifies a novel signalling integration hub where recycling endosomes orchestrate cellular behaviour. Therefore, targeting reciprocal priming over individual receptors may improve personalized therapies in breast and other cancers.

cell biology↗

bioRxiv2020

Targeting STAT3 signalling using stabilised sulforaphane (SFX-01) inhibits endocrine resistant stem-like cells in ER-positive breast cancer

PURPOSEEstrogen receptor (ER) positive breast cancer is frequently sensitive to endocrine therapy. Multiple mechanisms of endocrine therapy resistance have been identified, including cancer stem-like cell (CSC) activity. Sulforaphane (SFN) has previously been shown to target CSCs but its mechanism of action is unclear. Here we investigate SFX-01, a stabilised formulation of SFN, for its effects on breast CSC activity in ER+ preclinical models and to study its mechanism. EXPERIMENTAL DESIGNCSC activity was measured by mammosphere formation efficiency (MFE), aldehyde dehydrogenase (ALDH) activity, and tumor formation using patient samples and patient-derived xenograft (PDX) tumors treated with SFX-01 alone or in combination with tamoxifen or fulvestrant. Gene expression and SFN target proteins in treated samples were assessed. RESULTSSFX-01 reduced MFE of both ER+ primary and metastatic patient samples. Both tamoxifen and fulvestrant increased MFE and ALDH activity of PDX tumors, which was reversed by combination with SFX-01. SFX-01 significantly reduced tumor initiating cell frequency in secondary transplants at limiting dilution and reduced the formation of spontaneous lung micrometastases by PDX tumors in mice. Mechanistically, we establish that both tamoxifen and fulvestrant induce STAT3 phosphorylation. SFX-01 suppressed phospho-STAT3 and SFN directly bound STAT3 in patient and PDX samples. Analysis of ALDH+ cells from endocrine-resistant patient samples revealed activation of STAT3 target genes MUC1 and OSMR, which were inhibited by SFX-01 in patient samples. Increased expression of these genes after 3 months endocrine treatment of ER+ patients (n=68) predicted poor prognosis. CONCLUSIONSOur data establish the importance of STAT3 signaling in CSC-mediated resistance to endocrine therapy and the potential of SFX-01 for improving clinical outcomes in ER+ breast cancer.

cancer biology↗

bioRxiv2019

Targeting endometrial cancer stem cell activity with metformin is inhibited by patient-derived adipocyte-secreted factors

Translational relevanceEndometrial cancer stem cells are reported to drive metastasis and disease relapse and are resistant to commonly used chemotherapeutic drugs. Here, we characterise ALDHhigh and CD133+ve endometrial cancer stem cells with differing levels of cancer stem cell activity and stem cell and epithelial-mesenchymal transition (EMT) gene expression. Their proportions and activity are reduced following treatment with metformin at a lower concentration than that required to affect bulk tumour cell proliferation, establishing a selective and specific effect on endometrial cancer stem cells. We demonstrate that this effect is abrogated when endometrial cancer cells are pre-treated with adipocyte conditioned media, indicating adipokine-mediated resistance to metformin. These results support future studies of metformin as an adjuvant therapy to reduce the risk of disease recurrence but should also investigate the impact of obesity on treatment response. AbstractO_ST_ABSPurposeC_ST_ABSAdvanced endometrial cancer continues to have a poor prognosis due to limited treatment options, which may be further adversely impacted by obesity. Endometrial cancer stem cells have been reported to drive metastasis, chemotherapy resistance and disease relapse, but have yet to be fully characterised and no specific targeted therapies have been identified. Here, we describe the phenotype and genotype of aldehyde dehydrogenase high (ALDHhigh) and CD133+ve endometrial cancer stem cells and how adipocyte secreted mediators block the inhibitory effect of metformin on endometrial cancer stem cell activity. Experimental designIshikawa and Hec-1a cell lines were used to characterise ALDHhigh and CD133+ve endometrial cancer cells using flow cytometry, functional sphere assays and quantitative-Polymerase Chain Reaction. The comparative effect of metformin on endometrial cancer stem cell activity and bulk tumour cell proliferation was determined using an Aldefluor and cytotoxicity assay. The impact of adipocyte secreted mediators on metformin response was established using patient-derived conditioned media. ResultsALDHhigh cells demonstrated greater endometrial cancer stem cell activity than CD133+ve cells and had increased expression of stem cell and epithelial-mesenchymal transition genes. Treatment with 0.5-1mM metformin reduced the proportion and activity of both endometrial cancer stem cell populations (p[≤]0.05), without affecting cell viability. This effect was, however, inhibited by exposure to patient-derived adipocyte conditioned media. ConclusionsThese results indicate a selective and specific effect of metformin on endometrial cancer stem cell activity, which is blocked by adipocyte secreted mediators. Future studies of metformin as an adjuvant therapy in endometrial cancer should be adequately powered to investigate the influence of body mass on treatment response.

cancer biology↗

bioRxiv2019

The milk protein alpha-casein suppresses triple negative breast cancer stem cell activity via STAT and HIF-1alpha signalling pathways in breast cancer cells and fibroblasts

Triple negative breast cancer (TNBC) is the most lethal breast cancer subtype. Extended periods of lactation protect against breast cancer development, but the mechanisms underlying this protection are unknown. We examined the effects of the milk protein alpha-casein over expression in the triple negative MDA-MB-231 breast cancer cell line. The effects of recombinant alpha-casein added exogenously to MDA-MB-231 breast cancer cells, and immortalised human fibroblasts were also investigated. We used transcriptional reporters to understand the signalling pathways downstream of alpha-casein in breast cancer cells and these fibroblasts that were activated by breast cancer cells. To extend our findings to the clinical setting, we analysed public gene expression datasets to further understand the relevance of these signalling pathways in triple negative breast cancer cells and patient samples. Finally, we used small molecular inhibitors to target relevant pathways and highlight these as potential candidates for the treatment of TN breast cancer. High levels of alpha-casein gene expression were predictive of good prognosis across 263 TNBC patient tumour samples. Alpha-casein over expression or exogenous addition reduces cancer stem cell (CSC) activity. HIF-1alpha was identified to be a key downstream target of alpha-casein, in both breast cancer cells and activated fibroblasts, and STAT transcription factors to be upstream of HIF-1alpha. Interestingly, HIF-1alpha is regulated by STAT3 in breast cancer cells, but STAT1 is the regulator of HIF-1alpha in activated fibroblasts. In analysis of 573 TNBC patient samples, alpha-casein expression, inversely correlated to HIF-1alpha, STAT3 and STAT1. STAT1 and STAT3 inhibitors target HIF-1alpha signalling in activated fibroblasts and MDA-MB-231 breast cancer cells respectively, and also abrogate CSC activities. Our findings provide an explanation for the protective effects of lactation in TNBC. Clinical data correlates high alpha-casein expression with increased recurrence-free survival in TNBC patients. Mechanistically, alpha-casein reduces breast cancer stem cell activity in vitro, and STAT3 and STAT1 were identified as regulators of pro-tumorigenic HIF-1alpha signalling in breast cancer cells and fibroblasts respectively.

cancer biology↗