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Summary: Tumor self-seeding is a process whereby circulating tumor cells (CTCs) recolonize the primary tumor, which promotes tumor growth, angiogenesis, and invasion. However, the detailed nature and functions of tumor self-seeded cells (TSCs) have not been well defined due to challenges in tracking and isolating TSCs. Here, we report an accurate animal model using photoconvertible tagging to recapitulate the spontaneous process of tumor self-seeding and identify TSCs as a subpopulation of primary tumor cells with enhanced invasiveness and survival. We demonstrate transmembrane-4-L-six-family-1 (TM4SF1) as a marker of TSCs, which promotes migration, invasion, and anchorage-independent survival in cancer cells. By analyzing single-cell RNA sequencing datasets, we identify a potential TSC population with a metastatic profile in patients with cancer, which is detectable in early-stage disease and expands during cancer progression. In summary, we establish a framework to study TSCs and identify emerging cell targets with diagnostic, prognostic, or therapeutic potential in cancers.

Summary: Cancer cell populations comprise phenotypes distributed among the epithelial-mesenchymal (E-M) spectrum. However, it remains unclear which population-level processes give rise to the observed experimental distribution and dynamical changes in E-M heterogeneity, including (1) differential growth, (2) cell-state switching, and (3) population density-dependent growth or state-transition rates. Here, we analyze the necessity of these three processes in explaining the dynamics of E-M population distributions as observed in PMC42-LA and HCC38 breast cancer cells. We find that, while cell-state transition is necessary to reproduce experimental observations of dynamical changes in E-M fractions, including density-dependent growth interactions (cooperation or suppression) better explains the data. Further, our models predict that treatment of HCC38 cells with transforming growth factor β (TGF-β) signaling and Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/3) inhibitors enhances the rate of mesenchymal-epithelial transition (MET) instead of lowering that of E-M transition (EMT). Overall, our study identifies the population-level processes shaping the dynamics of spontaneous E-M heterogeneity in breast cancer cells.

Cold atmospheric plasma (CAP) holds promise as a cancer-specific treatment that selectively kills various types of malignant cells. We used CAP-activated media (PAM) to utilize a range of the generated short- and long-lived reactive species. Specific antibodies, small molecule inhibitors and CRISPR/Cas9 gene-editing approaches showed an essential role for receptor tyrosine kinases, especially epidermal growth factor (EGF) receptor, in mediating triple negative breast cancer (TNBC) cell responses to PAM. EGF also dramatically enhanced the sensitivity and specificity of PAM against TNBC cells. Site-specific phospho-EGFR analysis, signal transduction inhibitors and reconstitution of EGFR-depleted cells with EGFR-mutants confirmed the role of phospho-tyrosines 992/1173 and phospholipase C gamma signaling in up-regulating levels of reactive oxygen species above the apoptotic threshold. EGF-triggered EGFR activation enhanced the sensitivity and selectivity of PAM effects on TNBC cells. The proposed approach based on the synergy of CAP and EGFR-targeted therapy may provide new opportunities to improve the clinical management of TNBC.

Abstract Epithelial‐mesenchymal transition (EMT) is a reversible transcriptional program invoked by cancer cells to drive cancer progression. Transcription factor ZEB1 is a master regulator of EMT, driving disease recurrence in poor‐outcome triple negative breast cancers (TNBCs). Here, this work silences ZEB1 in TNBC models by CRISPR/dCas9‐mediated epigenetic editing, resulting in highly‐specific and nearly complete suppression of ZEB1 in vivo, accompanied by long‐lasting tumor inhibition. Integrated “omic” changes promoted by dCas9 linked to the KRAB domain (dCas9‐KRAB) enabled the discovery of a ZEB1‐dependent‐signature of 26 genes differentially‐expressed and ‐methylated, including the reactivation and enhanced chromatin accessibility in cell adhesion loci, outlining epigenetic reprogramming toward a more epithelial state. In the ZEB1 locus transcriptional silencing is associated with induction of locally‐spread heterochromatin, significant changes in DNA methylation at specific CpGs, gain of H3K9me3, and a near complete erasure of H3K4me3 in the ZEB1 promoter. Epigenetic shifts induced by ZEB1‐silencing are enriched in a subset of human breast tumors, illuminating a clinically‐relevant hybrid‐like state. Thus, the synthetic epi‐silencing of ZEB1 induces stable “lock‐in” epigenetic reprogramming of mesenchymal tumors associated with a distinct and stable epigenetic landscape. This work outlines epigenome‐engineering approaches for reversing EMT and customizable precision molecular oncology approaches for targeting poor outcome breast cancers.

Abstract Epithelial–mesenchymal transition (EMT) is a dynamic and complex cellular process that is known to be hijacked by cancer cells to facilitate invasion, metastasis and therapeutic resistance. Several quantitative measures to assess the interplay between EMT and cancer progression are available, based on large scale genome and transcriptome data. However, these large scale multi-omics studies have repeatedly illustrated a lack of correlation in mRNA and protein abundances that may be influenced by diverse post-translational regulation. Hence, it is imperative to understand how changes in the EMT proteome are associated with the process of oncogenic transformation. To this effect, we developed a parallel reaction monitoring-based targeted proteomics method for quantifying abundances of EMT-associated proteins across cancer cell lines. Our study revealed that quantitative measurement of EMT proteome which enabled a more accurate assessment than transcriptomics data and revealed specific discrepancies against a backdrop of generally strong concordance between proteomic and transcriptomic data. We further demonstrated that changes in our EMT proteome panel might play a role in tumor transformation across cancer types. In future, this EMT panel assay has the potential to be used for clinical samples to guide treatment choices and to congregate functional information for the development and advancing novel therapeutics.

Abstract Background Triple-negative breast cancers (TNBC) have a relatively poor prognosis and responses to targeted therapies. Between 25 and 39% of TNBCs are claudin-low, a poorly differentiated subtype enriched for mesenchymal, stem cell and mitogen-activated signaling pathways. We investigated the role of the cell-surface co-receptor NRP1 in the biology of claudin-low TNBC. Methods The clinical prognostic value of NRP1 was determined by Kaplan–Meier analysis. GSVA analysis of METABRIC and Oslo2 transcriptomics datasets was used to correlate NRP1 expression with claudin-low gene signature scores. NRP1 siRNA knockdown was performed in MDA-MB-231, BT-549, SUM159 and Hs578T claudin-low cells and proliferation and viability measured by live cell imaging and DNA quantification. In SUM159 orthotopic xenograft models using NSG mice, NRP1 was suppressed by shRNA knockdown or systemic treatment with the NRP1-targeted monoclonal antibody Vesencumab. NRP1-mediated signaling pathways were interrogated by protein array and Western blotting. Results High NRP1 expression was associated with shorter relapse- and metastasis-free survival specifically in ER-negative BrCa cohorts. NRP1 was over-expressed specifically in claudin-low clinical samples and cell lines, and NRP1 knockdown reduced proliferation of claudin-low cells and prolonged survival in a claudin-low orthotopic xenograft model. NRP1 inhibition suppressed expression of the mesenchymal and stem cell markers ZEB1 and ITGA6, respectively, compromised spheroid-initiating capacity and exerted potent anti-tumor effects on claudin-low orthotopic xenografts (12.8-fold reduction in endpoint tumor volume). NRP1 was required to maintain maximal RAS/MAPK signaling via EGFR and PDGFR, a hallmark of claudin-low tumors. Conclusions These data implicate NRP1 in the aggressive phenotype of claudin-low breast cancer and offer a novel targeted therapeutic approach to this poor prognosis subtype.

Abstract Background Breast cancers acquire aggressive capabilities via epithelial to mesenchymal transition (EMT), in which various integrins/integrin-linked kinase signalling are upregulated. Methods We investigated this in two patient-derived xenografts (PDXs) developed from breast-to-bone metastases, and its functional significance in a breast cancer cell line system. ED03 and EDW01 PDXs were grown subcutaneously in immunocompromised SCID mice through 11 passages and 7 passages, respectively. Tumour tissue was assessed using immunohistochemistry (IHC) for oestrogen receptor (ER)-alpha, E-cadherin, vimentin, Twist1, beta-catenin, P120-RasGAP, CD44, CD24 and Ki67, and RT-qPCR of EMT-related factors (CDH1, VIM, CD44, CD24), integrins beta 1 (ITGB1), alpha 2 (ITGA2) and ILK. Integrin and ILK expression in epidermal growth factor (EGF)-induced EMT of the PMC42-ET breast cancer cell line was assessed by RT-qPCR and Western blotting, as were the effects of their transient knockdown via small interfering RNA +/− EGF. Cell migration, changes in cell morphology and adhesion of siRNA-transfected PMC42-ET cells to various extracellular matrix (ECM) substrates was assessed. Results The ED03 (ER+/PR−/HER2−/lobular) and EDW01 (ER+/PR−/HER2−/ductal) PDXs were both classified as molecular subtype luminal A. ED03 xenografts exhibited mutated E-cadherin with minimal expression, but remained vimentin-negative across all passages. In EDW01, the hypoxic indicator gene CAIX and Twist1 were co-ordinately upregulated at passages 4–5, corresponding with a decrease in E-cadherin. At passages 6–7, VIM was upregulated along with ITGB1 and ITGA2, consistent with an increasing EMT. The ED03 PDX displayed minimal change over passages in mice, for all genes examined. ILK, ITGB1 and ITGA2 mRNAs were also increased in the EGF-induced EMT of PMC42-ET cells (in which CDH1 was downregulated) although siRNA against these targets revealed that this induction was not necessary for the observed EMT. However, their knockdown significantly reduced EMT-associated adhesion and Transwell migration. Conclusion Our data suggest that despite an increase in ITGA2 and ITGB1 gene expression in the EMT exhibited by EDW01 PDX over multiple generations, this pathway may not necessarily drive the EMT process.

Castrate-resistant prostate cancer (CRPC) is the lethal form of prostate cancer. Epithelial mesenchymal plasticity (EMP) has been associated with disease progression to CRPC, and prostate cancer therapies targeting the androgen signalling axis, including androgen deprivation therapy (ADT), promote EMP. We explored effects of castration on EMP in the tumours and circulating tumour cells (CTCs) of patient-derived xenograft (PDX)-bearing castrated mice using human-specific RT-qPCR assays and immunocytochemistry. Expression of prostate epithelial cell marker KLK3 was below detection in most tumours from castrated mice (62%, 23/37 mice), consistent with its known up-regulation by androgens. Endpoint tumour size after castration varied significantly in a PDX model-specific pattern; while most tumours were castration-sensitive (BM18, LuCaP70), the majority of LuCaP105 tumours continued to grow following castration. By contrast, LuCaP96 PDX showed a mixed response to castration. CTCs were detected in 33% of LuCaP105, 43% of BM18, 47% of LuCaP70, and 54% of LuCaP96 castrated mice using RPL32 mRNA measurement in plasma. When present, CTC numbers estimated using human RPL32 expression ranged from 1 to 458 CTCs per ml blood, similar to our previous observations in non-castrated mice. In contrast to their non-castrated counterparts, there was no relationship between tumour size and CTC burden in castrated mice. Unsupervised hierarchical clustering of the gene expression profiles of CTCs collected from castrated and non-castrated mice revealed distinct CTC sub-groups within the pooled population that were classified as having mesenchymal, epithelial, or EMP hybrid gene expression profiles. The epithelial signature was only found in CTCs from non-castrated mice. Hybrid and mesenchymal signatures were detected in CTCs from both castrated and non-castrated mice, with an emphasis towards mesenchymal phenotypes in castrated mice. Post-castration serum PSA levels were either below detection or very low for all the CTC positive samples highlighting the potential usefulness of CTCs for disease monitoring after androgen ablation therapy. In summary, our study of castration effects on prostate cancer PDX CTCs showed that CTCs were often detected in the castrate setting, even in mice with no palpable tumours, and demonstrated the superior ability of CTCs to reveal residual disease over the conventional clinical biomarker serum PSA.

Circulating tumour cells (CTCs) are shed into the bloodstream from both primary and secondary tumours and provide a non-invasive means to study tumor progression and response to treatment. Assessment of ribonucleic acid (RNA) and monitoring dynamic changes in gene expression profiles of CTCs extends their clinical and prognostic power and establish their role in guiding treatment. Among these methods, droplet digital (RT-ddPCR) technique provides a high sensitivity and detectibility of CTCs. RNA-sequencing (RNAseq) is the most comprehensive method, that would allow the simultaneous measurement of a large number of genes and theoretically the whole transcriptome. Since CTCs are heterogeneous in nature, single cell RNAseq methods are very valuable in assessing population dynamics and functional states of CTCs. While RNA in situ hybridization (RNA-ISH) is used relatively less frequently, it also allows for the assessment of expression of multiple genes within individual CTCs. Epithelial to Mesenchymal Transition (EMT) or Plasticity (EMP) is a major contributor to metastasis, providing a mechanism to allow cells to become migratory and invasive, and to survive in the bloodstream. Monitoring CTCs undergoing EMT may lead to improvement in their prognostic and predictive power. Here, we review various RNA analysis of CTCs and those that undergo EMT and their application in diagnosis, prognosis and treatment of cancers.

3D organoid model technologies have led to the development of innovative tools for cancer precision medicine. Yet, the gold standard culture system (Matrigel®) lacks the ability for extensive biophysical manipulation needed to model various cancer microenvironments and has inherent batch-to-batch variability. Tunable hydrogel matrices provide enhanced capability for drug testing in breast cancer (BCa), by better mimicking key physicochemical characteristics of this disease’s extracellular matrix. Here, we encapsulated patient-derived breast cancer cells in bioprinted polyethylene glycol-derived hydrogels (PEG), functionalized with adhesion peptides (RGD, GFOGER and DYIGSR) and gelatin-derived hydrogels (gelatin methacryloyl; GelMA and thiolated-gelatin crosslinked with PEG-4MAL; GelSH). Within ranges of BCa stiffnesses (1–6 kPa), GelMA, GelSH and PEG-based hydrogels successfully supported the growth and organoid formation of HR+,−/HER2+,− primary cancer cells for at least 2–3 weeks, with superior organoid formation within the GelSH biomaterial (up to 268% growth after 15 days). BCa organoids responded to doxorubicin, EP31670 and paclitaxel treatments with increased IC50 concentrations on organoids compared to 2D cultures, and highest IC50 for organoids in GelSH. Cell viability after doxorubicin treatment (1 µM) remained >2-fold higher in the 3D gels compared to 2D and doxorubicin/paclitaxel (both 5 µM) were ~2.75–3-fold less potent in GelSH compared to PEG hydrogels. The data demonstrate the potential of hydrogel matrices as easy-to-use and effective preclinical tools for therapy assessment in patient-derived breast cancer organoids.

Precision medicine approaches that inform clinical management of individuals with cancer are progressively advancing. Patient-derived explants (PDEs) provide a patient-proximal ex vivo platform that can be used to assess sensitivity to standard of care (SOC) therapies and novel agents. PDEs have several advantages as a patient-proximal model compared to current preclinical models, as they maintain the phenotype and microenvironment of the individual tumor. However, the longevity of PDEs is not compatible with the timeframe required to incorporate candidate therapeutic options identified by whole exome sequencing (WES) of the patient’s tumor. This review investigates how PDE longevity varies across tumor streams and how this is influenced by tissue preparation. Improving longevity of PDEs will enable individualized therapeutics testing, and thus contribute to improving outcomes for people with cancer.

Cancer cells are more vulnerable to abnormal redox fluctuations due to their imbalanced antioxidant system, where cell surface receptors sense stress and trigger intracellular signal relay. As canonical targets of many targeted therapies, cell receptors sensitize the cells to specific drugs. On the other hand, cell target mutations are commonly associated with drug resistance. Thus, exploring effective therapeutics targeting diverse cell receptors may open new clinical avenues against aggressive cancers. This paper uses focused case studies to reveal the intrinsic relationship between the cell receptors of different categories and the primary cancer hallmarks that are associated with the responses to external or internal redox perturbations. Cold atmospheric plasma (CAP) is examined as a promising redox modulation medium and highly selective anti-cancer therapeutic modality featuring dynamically varying receptor targets and minimized drug resistance against aggressive cancers.

The extracellular matrix (ECM) provides cues to direct mammogenesis, tumourigenesis and metastatic processes. Over the past several decades, two-dimensional (2D) culture models have been invaluable in furthering our understanding of the tumor microenvironment (TME), however, they still do not accurately emulate the associated biological complexities. In contrast, three-dimensional (3D) culture models provide a more physiologically relevant platform to study relevant physicochemical signals, stromal-epithelial cell interactions, vascular and immune components, and cell-ECM interactions in the human breast microenvironment. A common thread that may weave these multiple interactions are the proteoglycans (PGs), a prominent family of molecules in breast tissue. This review will discuss how these PGs contribute to the breast cancer TME and provide a summary of the traditional and emerging technologies that have been utilized to better understand the role of PGs during malignant transformation. Furthermore, this review will emphasize the differences that PGs exhibit between normal tissues and tumor ECM, providing a rationale for the investigation of underexplored roles of PGs in breast cancer progression using state-of-the-art 3D culture models.

Mammographic density (MD) is a strong and independent factor for breast cancer (BC) risk and is increasingly associated with BC progression. We have previously shown in mice that high MD, which is characterized by the preponderance of a fibrous stroma, facilitates BC xenograft growth and metastasis. This stroma is rich in extracellular matrix (ECM) factors, including heparan sulfate proteoglycans (HSPGs), such as the BC-associated syndecan-1 (SDC1). These proteoglycans tether growth factors, which are released by heparanase (HPSE). MD is positively associated with estrogen exposure and, in cell models, estrogen has been implicated in the upregulation of HPSE, the activity of which promotes SDC expression. Herein we describe a novel measurement approach (single-sided NMR) using a patient-derived explant (PDE) model of normal human (female) mammary tissue cultured ex vivo to investigate the role(s) of HPSE and SDC1 on MD. Relative HSPG gene and protein analyses determined in patient-paired high vs. low MD tissues identified SDC1 and SDC4 as potential mediators of MD. Using the PDE model we demonstrate that HPSE promotes SDC1 rather than SDC4 expression and cleavage, leading to increased MD. In this model system, synstatin (SSTN), an SDC1 inhibitory peptide designed to decouple SDC1-ITGαvβ3 parallel collagen alignment, reduced the abundance of fibrillar collagen as assessed by picrosirius red viewed under polarized light, and reduced MD. Our results reveal a potential role for HPSE in maintaining MD via its direct regulation of SDC1, which in turn physically tethers collagen into aligned fibers characteristic of MD. We propose that inhibitors of HPSE and/or SDC1 may afford an opportunity to reduce MD in high BC risk individuals and reduce MD-associated BC progression in conjunction with established BC therapies.

Abstract The development of clustered regularly interspaced short palindromic repeats (CRISPR) has spurred a successive wave of genome‐engineering following zinc finger nucleases and transcription activator‐like effector nucleases, and made gene‐editing a promising strategy in the prevention and treatment of genetic diseases. However, gene‐editing is not widely adopted in clinics due to some technical issues that challenge its safety and efficacy, and the lack of appropriate clinical regulations allowing them to advance toward improved human health without impinging on human ethics. By systematically examining the oncological applications of gene‐editing tools and critical factors challenging their medical translation, genome‐editing has substantial contributions to cancer driver gene discovery, tumor cell epigenome normalization, targeted delivery, cancer animal model establishment, and cancer immunotherapy and prevention in clinics. Gene‐editing tools, epitomized by CRISPR, are predicted to represent a promising strategy toward the precise control of cancer initiation and development. However, some technical problems and ethical concerns are serious issues that need to be appropriately addressed before CRISPR can be incorporated into the next generation of molecular precision medicine. In this light, new technical developments to limit off‐target effects are discussed herein, and the use of gene‐editing approaches for treating otherwise incurable cancers is brought into focus.

Tumor cells demonstrate substantial plasticity in their genotypic and phenotypic characteristics. Epithelial-mesenchymal plasticity (EMP) can be characterized into dynamic intermediate states and can be orchestrated by many factors, either intercellularly via epigenetic reprograming, or extracellularly via growth factors, inflammation and/or hypoxia generated by the tumor stromal microenvironment. EMP has the capability to alter phenotype and produce heterogeneity, and thus by changing the whole cancer landscape can attenuate oncogenic signaling networks, invoke anti-apoptotic features, defend against chemotherapeutics and reprogram angiogenic and immune recognition functions. We discuss here the role of phenotypic plasticity in tumor initiation, progression and metastasis and provide an update of the modalities utilized for the molecular characterization of the EMT states and attributes of cellular behavior, including cellular metabolism, in the context of EMP. We also summarize recent findings in dynamic EMP studies that provide new insights into the phenotypic plasticity of EMP flux in cancer and propose therapeutic strategies to impede the metastatic outgrowth of phenotypically heterogeneous tumors.

Phenotypic heterogeneity is a hallmark of aggressive cancer behaviour and a clinical challenge. Despite much characterisation of this heterogeneity at a multi-omics level in many cancers, we have a limited understanding of how this heterogeneity emerges spontaneously in an isogenic cell population. Some longitudinal observations of dynamics in epithelial-mesenchymal heterogeneity, a canonical example of phenotypic heterogeneity, have offered us opportunities to quantify the rates of phenotypic switching that may drive such heterogeneity. Here, we offer a mathematical modeling framework that explains the salient features of population dynamics noted in PMC42-LA cells: (a) predominance of EpCAMhigh subpopulation, (b) re-establishment of parental distributions from the EpCAMhigh and EpCAMlow subpopulations, and (c) enhanced heterogeneity in clonal populations established from individual cells. Our framework proposes that fluctuations or noise in content duplication and partitioning of SNAIL—an EMT-inducing transcription factor—during cell division can explain spontaneous phenotypic switching and consequent dynamic heterogeneity in PMC42-LA cells observed experimentally at both single-cell and bulk level analysis. Together, we propose that asymmetric cell division can be a potential mechanism for phenotypic heterogeneity.

Abstract Background Epidemiological studies have consistently shown that increased mammographic density (MD) is a strong risk factor for breast cancer. We previously observed an elevated number of vimentin+/CD45+ leukocytes in high MD (HMD) epithelium. In the present study, we aimed to investigate the subtypes of immune cell infiltrates in HMD and low MD (LMD) breast tissue. Methods Fifty-four women undergoing prophylactic mastectomy at Peter MacCallum Cancer Centre or St. Vincent’s Hospital were enrolled. Upon completion of mastectomy, HMD and LMD areas were resected under radiological guidance in collaboration with BreastScreen Victoria and were subsequently fixed, processed, and sectioned. Fifteen paired HMD and LMD specimens were further selected according to their fibroglandular characteristics (reasonable amount [> 20%] of tissue per block on H&E stains) for subsequent IHC analysis of immune cell infiltration. Results Overall, immune cell infiltrates were predominantly present in breast ducts and lobules rather than in the stroma, with CD68+ macrophages and CD20+ B lymphocytes also surrounding the vasculature. Macrophages, dendritic cells (DCs), B lymphocytes, and programmed cell death protein 1 (PD-1) expression were significantly increased in HMD epithelium compared with LMD. Moreover, significantly higher levels of DCs, CD4+ T cells, and PD-1 were also observed in HMD stroma than in LMD stroma. The increased expression of interleukin (IL)-6 and IL-4, with unaltered interferon-γ, indicate a proinflammatory microenvironment. Conclusions Our work indicates that the immune system may be activated very early in breast cancer development and may in part underpin the breast cancer risk associated with HMD.

Abstract One of the hallmarks of the tumour microenvironment is hypoxia resulting from increased oxygen consumption by proliferative cancer cells and altered vasculature. Hypoxic tension initiates various cellular signals and can drive epithelial to mesenchymal transition (EMT), a process important in cancer progression. In this study, using the antioxidant N-acetylcysteine (NAC), we show that hypoxia-induced reactive oxygen species (ROS) in MDA-MB-468 breast cancer cells, selectively regulate hypoxia-induced increases in N-cadherin and SERPINE1, two proteins involved in cell adhesion. Treatment of cells with NAC also attenuated hypoxia-mediated activation of EGFR, but did not have any effect on hypoxia-mediated induction of HIF1α. Exogenous hydrogen peroxide phenocopied the effects of hypoxia on N-cadherin and SERPINE1 expression and EGFR activation, suggesting its possible involvement in these hypoxia-mediated events. Reflective of their effect on cell adhesion proteins and EGFR (associated with migratory phenotypes), NAC also reduced cell migration under hypoxic conditions, a crucial event in metastasis. Our findings suggest a selective role for redox signalling in the regulation of specific components of the responses to hypoxia and induction of EMT in breast cancer cells. This study provides new evidence supporting the potential of targeting ROS as a therapeutic strategy for the control of breast cancer metastasis.

Aims/Hypothesis: Previous studies have associated serum ferritin levels with type 2 diabetes, so it's well recognised that there is a subset of patients with type 2 diabetes and also with elevated serum ferritin. The aim of this pilot study was to test whether venesection is a viable treatment option for patients with type 2 diabetes. Methods: Four cases with elevated serum ferritin and type 2 diabetes were subjected to monthly venesection for 3–9 months, and monitored 3 monthly for serum ferritin and glycated haemoglobin (HbA1c). Results: In all four cases, serum ferritin was dramatically reduced, and the HbA1c was reduced in all but one case, which was a diet-only controlled patient with diabetes and had near normal HbA1c. In two cases, HbA1c was reduced by 2–3% units (22–33 mmol/mol), making it superior to any current medication. Conclusions/Interpretation: Venesection was used here successfully with oral pharmacotherapy, thereby avoiding the use of insulin. Findings of this study may provide clinicians a powerful new tool to intervene in the pathological process of type 2 diabetes by a very simple manoeuvre - venesection. These results need to be repeated and validated in a larger trial as a priority.

Abstract Drug resistance continues to be a major barrier to the delivery of curative therapies in cancer. Historically, drug resistance has been associated with over‐expression of drug transporters, changes in drug kinetics or amplification of drug targets. However, the emergence of resistance in patients treated with new‐targeted therapies has provided new insight into the complexities underlying cancer drug resistance. Recent data now implicate intratumoural heterogeneity as a major driver of drug resistance. Single cell sequencing studies that identified multiple genetically distinct variants within human tumours clearly demonstrate the heterogeneous nature of human tumours. The major contributors to intratumoural heterogeneity are (i) genetic variation, (ii) stochastic processes, (iii) the microenvironment and (iv) cell and tissue plasticity. Each of these factors impacts on drug sensitivity. To deliver curative therapies to patients, modification of current therapeutic strategies to include methods that estimate intratumoural heterogeneity and plasticity will be essential.