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Most cancer deaths are due to metastasis, and epithelial-to-mesenchymal transition (EMT) plays a central role in driving cancer cell metastasis. EMT is induced by different stimuli, leading to different signaling patterns and therapeutic responses. TGFβ is one of the best-studied drivers of EMT, and many drugs are available to target this signaling pathway. A comprehensive bioinformatics approach was employed to derive a signature for TGFβ-induced EMT which can be used to score TGFβ-driven EMT in cells and clinical specimens. Considering this signature in pan-cancer cell and tumor datasets, a number of cell lines (including basal B breast cancer and cancers of the central nervous system) show evidence for TGFβ-driven EMT and carry a low mutational burden across the TGFβ signaling pathway. Furthermore, significant variation is observed in the response of high scoring cell lines to some common cancer drugs. Finally, this signature was applied to pan-cancer data from The Cancer Genome Atlas to identify tumor types with evidence of TGFβ-induced EMT. Tumor types with high scores showed significantly lower survival rates than those with low scores and also carry a lower mutational burden in the TGFβ pathway. The current transcriptomic signature demonstrates reproducible results across independent cell line and cancer datasets and identifies samples with strong mesenchymal phenotypes likely to be driven by TGFβ.Implications: The TGFβ-induced EMT signature may be useful to identify patients with mesenchymal-like tumors who could benefit from targeted therapeutics to inhibit promesenchymal TGFβ signaling and disrupt the metastatic cascade. Mol Cancer Res; 15(5); 619–31. ©2017 AACR.

Expanded and additional figures supporting this manuscript.

Detailed description of computational methods used in this manuscript.

Expanded and additional tables supporting this manuscript.

Epithelial–mesenchymal transition (EMT) is prominent in circulating tumor cells (CTC), but how it influences metastatic spread in this setting is obscure. Insofar as blood provides a specific microenvironment for tumor cells, we explored a potential link between EMT and coagulation that may provide EMT-positive CTCs with enhanced colonizing properties. Here we report that EMT induces tissue factor (TF), a major cell-associated initiator of coagulation and related procoagulant properties in the blood. TF blockade by antibody or shRNA diminished the procoagulant activity of EMT-positive cells, confirming a functional role for TF in these processes. Silencing the EMT transcription factor ZEB1 inhibited both EMT-associated TF expression and coagulant activity, further strengthening the link between EMT and coagulation. Accordingly, EMT-positive cells exhibited a higher persistance/survival in the lungs of mice colonized after intravenous injection, a feature diminished by TF or ZEB1 silencing. In tumor cells with limited metastatic capability, enforcing expression of the EMT transcription factor Snail increased TF, coagulant properties, and early metastasis. Clinically, we identified a subpopulation of CTC expressing vimentin and TF in the blood of metastatic breast cancer patients consistent with our observations. Overall, our findings define a novel EMT–TF regulatory axis that triggers local activation of coagulation pathways to support metastatic colonization of EMT-positive CTCs. Cancer Res; 76(14); 4270–82. ©2016 AACR.

Supplementary Table 2 from Mesenchymal-to-Epithelial Transition Facilitates Bladder Cancer Metastasis: Role of Fibroblast Growth Factor Receptor-2

Epithelial-to-mesenchymal transition (EMT) increases cell migration and invasion, and facilitates metastasis in multiple carcinoma types, but belies epithelial similarities between primary and secondary tumors. This study addresses the importance of mesenchymal-to-epithelial transition (MET) in the formation of clinically significant metastasis. The previously described bladder carcinoma TSU-Pr1 (T24) progression series of cell lines selected in vivo for increasing metastatic ability following systemic seeding was used in this study. It was found that the more metastatic sublines had acquired epithelial characteristics. Epithelial and mesenchymal phenotypes were confirmed in the TSU-Pr1 series by cytoskeletal and morphologic analysis, and by performance in a panel of in vitro assays. Metastatic ability was examined following inoculation at various sites. Epithelial characteristics associated with dramatically increased bone and soft tissue colonization after intracardiac or intratibial injection. In contrast, the more epithelial sublines showed decreased lung metastases following orthotopic inoculation, supporting the concept that EMT is important for the escape of tumor cells from the primary tumor. We confirmed the overexpression of the IIIc subtype of multiple fibroblast growth factor receptors (FGFR) through the TSU-Pr1 series, and targeted abrogation of FGFR2IIIc reversed the MET and associated functionality in this system and increased survival following in vivo inoculation in severe combined immunodeficient mice. This model is the first to specifically model steps of the latter part of the metastatic cascade in isogenic cell lines, and confirms the suspected role of MET in secondary tumor growth. (Cancer Res 2006; 66(23): 11271-8)

Supplementary Table 1 from Mesenchymal-to-Epithelial Transition Facilitates Bladder Cancer Metastasis: Role of Fibroblast Growth Factor Receptor-2

Supplementary Materials and Methods

Supplementary Table 1: list of siRNA used. Supplementary Table 2: list of primers used. Supplementary Table 3: list of antibodies used in different applications. Supplementary Table 4: list of patients and donors information

Supplementary Table 3 from Mesenchymal-to-Epithelial Transition Facilitates Bladder Cancer Metastasis: Role of Fibroblast Growth Factor Receptor-2

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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.

(1) Background: Epithelial&ndash, mesenchymal plasticity (EMP) is a dynamic process whereby epithelial carcinoma cells reversibly acquire morphological and invasive characteristics typical of mesenchymal cells. Identifying the methylation differences between epithelial and mesenchymal states may assist in the identification of optimal DNA methylation biomarkers for the blood-based monitoring of cancer. (2) Methods: Methylation-sensitive high-resolution melting (MS-HRM) was used to examine the promoter methylation status of a panel of established and novel markers in a range of breast cancer cell lines spanning the epithelial&ndash, mesenchymal spectrum. Pyrosequencing was used to validate the MS-HRM results. (3) Results: VIM, DKK3, and CRABP1 were methylated in the majority of epithelial breast cancer cell lines, while methylation of GRHL2, MIR200C, and CDH1 was restricted to mesenchymal cell lines. Some markers that have been used to assess minimal residual disease such as AKR1B1 and APC methylation proved to be specific for epithelial breast cell lines. However, RASSF1A, RAR&beta, TWIST1, and SFRP2 methylation was seen in both epithelial and mesenchymal cell lines, supporting their suitability for a multimarker panel. (4) Conclusions: Profiling DNA methylation shows a distinction between epithelial and mesenchymal phenotypes. Understanding how DNA methylation varies between epithelial and mesenchymal phenotypes may lead to more rational selection of methylation-based biomarkers for circulating tumour DNA analysis.

Regions of high mammographic density (MD) in the breast are characterised by a proteoglycan (PG)-rich fibrous stroma, where PGs mediate aligned collagen fibrils to control tissue stiffness and hence the response to mechanical forces. Literature is accumulating to support the notion that mechanical stiffness may drive PG synthesis in the breast contributing to MD. We review emerging patterns in MD and other biological settings, of a positive feedback cycle of force promoting PG synthesis, such as in articular cartilage, due to increased pressure on weight bearing joints. Furthermore, we present evidence to suggest a pro-tumorigenic effect of increased mechanical force on epithelial cells in contexts where PG-mediated, aligned collagen fibrous tissue abounds, with implications for breast cancer development attributable to high MD. Finally, we summarise means through which this positive feedback mechanism of PG synthesis may be intercepted to reduce mechanical force within tissues and thus reduce disease burden.

Oral cavity cancer (OCC) is the predominant subtype of head and neck cancer (HNC) and has up to 50% mortality. Genome-wide microRNA (miR) sequencing data indicates overexpression of miR-9-5p in HNC tumours, however, the biological role of miR-9-5p in OCC is complex; it can either act as a tumour suppressor or an oncomir, regulating many target genes at the post-transcriptional level. We have investigated the overexpression of miR-9-5p in three OCC cell lines. We have evaluated its expression levels and Galectin-3 as potential biomarkers in saliva samples collected from controls and OCC patients. We found that over expression of miR-9-5p in OCC cell lines resulted in a significant reduction in cell proliferation and migration, and an increase in apoptosis, which was paralleled by an increase in Galectin-3 secretion and export of Galectin-3 protein. Our data are consistent with miR-9-5p being a modulator of Galectin-3 via the AKT/γ-catenin pathway. In addition, the positive correlation between the levels of miR-9-5p expression and secreted Galectin-3 in saliva reflects a similar relationship in vivo, and supports the utility of their integrative evaluation in OCC. Our findings indicate that both miR-9-5p and Galectin-3 are critical biomolecules in the progression of OCC.

BackgroundBreast cancers acquire aggressive capabilities via epithelial to mesenchymal transition (EMT), in which various integrins/integrin-linked kinase signalling are upregulated.MethodsWe 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) andILK. Integrin andILKexpression 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.ResultsThe 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,VIMwas upregulated along withITGB1andITGA2, consistent with an increasing EMT. The ED03 PDX displayed minimal change over passages in mice, for all genes examined.ILK,ITGB1andITGA2mRNAs were also increased in the EGF-induced EMT of PMC42-ET cells (in whichCDH1was 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.ConclusionOur data suggest that despite an increase inITGA2andITGB1gene expression in the EMT exhibited by EDW01 PDX over multiple generations, this pathway may not necessarily drive the EMT process.

Mammographic Density (MD) is the degree of radio-opacity of the breast in an X-ray mammogram. It is determined by the Fibroglandular: Adipose tissue ratio. MD has major implications in breast cancer risk and breast cancer chemoprevention. This study aimed to investigate the feasibility of accurate, low-cost quantification of MD in vivo without ionising radiation. We used single-sided portable nuclear magnetic resonance ("Portable NMR") due to its low cost and the absence of radiation-related safety concerns. Fifteen (N = 15) healthy female volunteers were selected for the study and underwent an imaging routine consisting of 2D X-ray mammography, quantitative breast 3T MRI (Dixon and T

Elevated levels of pregnancy-associated plasma protein-A (PAPP-A) have been implicated in the pathogenesis of various malignancies, including breast cancers. Breast cancer is one of the most frequent carcinomas and is the second most common cancer type detected in women of child-bearing age. Throughout pregnancy PAPP-A is produced and secreted by the placental syncytiotrophoblast cells; co-incidentally pregnancy-associated breast cancers often have an aggressive clinical course. The components of the PAPP-A/IGF axis was assessed in a panel of breast cancer cell lines. Using neutralising antibodies the impact of PAPP-A/IGF axis on cell motility was evaluated. PAPP-A was expressed in four of the twelve breast cancer cell lines tested. Blocking PAPP-A and IGFBP4 with neutralising antibodies significantly decreased motiliy of MDA-MB-231 cells. Upregulation of PAPP-A expression in breast tumours resulted in a trend towards worse overall survival. Notably, PAPP-A expression also positively correlated with epithelial-to-mesenchymal transition markers. In conclusion, these results indicate that PAPP-A plays an important role in breast cancer progression and it may be a promising therapeutic target in breast cancer.

The Ca2+ signal is essential in both hypoxia- and epidermal growth factor (EGF)-mediated epithelial to mesenchymal transition (EMT) in MDA-MB-468 breast cancer cells. This finding suggests that Ca2+-permeable ion channels participate in the induction of expression of some mesenchymal markers such as vimentin. However, the ion channels involved in vimentin expression induction have not been fully characterized. This work sought to define how differential modulation of the calcium signal effects the induction of vimentin and the Ca2+ influx pathways involved. We identified that the intracellular Ca2+ chelator EGTA-AM, cytochalasin D (a modulator of cytoskeletal dynamics and cell morphology), and the sarco/endoplasmic reticulum ATPase inhibitor thapsigargin are all inducers of vimentin in MDA-MB-468 breast cancer cells. EGTA-AM- and thapsigargin-mediated induction of vimentin expression in MDA-MB-468 cells involves store-operated Ca2+ entry, as evidenced by sensitivity to silencing of the molecular components of this pathway, STIM1 and ORAI1. In stark contrast, cytochalasin D-mediated vimentin induction was insensitive to silencing of ORAI1, despite sensitivity to silencing of its canonical activator the endoplasmic reticulum Ca2+ sensor STIM1. Cytochalasin D-mediated vimentin induction was, however, sensitive to silencing of another reported STIM1 target, TRPC1. Subsequent studies identified that EGTA-AM-induced vimentin expression also partially involved a TRPC1-dependent pathway. These studies define a complex interplay between vimentin expression in this model and the specific Ca2+-permeable ion channels involved. The complexity in the engagement of different Ca2+ influx pathways that regulate vimentin induction are opportunities but also potential challenges in targeting Ca2+ signaling to block EMT in cancer cells. Our findings further highlight the need to identify potential indispensable ion channels that can regulate induction of specific mesenchymal markers via different stimuli.

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.

Background At present, immunotherapy has become an established treatment for cancer. Recently, we have realized that tumor metabolism has a huge impact on the shaping of immune reactivity in the tumor microenvironment (TME). Exploration of the tumor metabolic and immunocellular response reveals metabolic vulnerability as a therapeutic window for intervention to enhance immunotherapy. The clinical significance of glycolysis and its role in tumor-immune evolution have not been fully explored. Understanding the relationships between glycolysis, TME evolution and disease progression is of great significance for optimizing immunotherapy of liver cancer. Methods A glycolysis-related biomarker signature was initially identified in transcriptomic data within The Cancer Genome Atlas (TCGA, n=424). Predicted overall survival (OS) for the gene signature in the GSE54236 dataset (n=161) and our HCC cohort (n=132) was subsequently independently verified. The relative number of immune cells in the microenvironment was calculated using CIBERSORT. CCK-8, IHC, Transwell and Seahorse were used to assess the effects of a 3-gene signature on the malignant phenotype of liver cancer. Results A 3-gene signature of glycolysis significantly associated with poor OS was identified. Based on a multi-omics research strategy, we found the 3-gene signature was closely related to the evolution of liver cancer TME and disease progression, and was associated with liver cancer mutation load and immune evolution. High-risk patients have inhibition and depletion of the immune TME and immune escape through antigen presentation inhibition. Remarkably, the 3-gene signature can predict the clinical outcomes of HCC patients with different clinical subtypes. Conclusions Our investigation of the 3-gene signature provided evidence for tumor metabolism-immune co-evolution along HCC progression. The 3-gene signature for stratification of liver cancer risk has robust predictive power, both at the RNA and protein levels.

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.

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.

Metastasis is responsible for more than 90% of cancer related deaths. Bone is one of the most frequent sites of metastasis from various primary carcinomas, especially breast and prostate. Therefore, understanding the molecular mechanisms underlying bone metastasis is a major focus of research. The polarized epithelial tumor cells undergo epithelial to mesenchymal transition (EMT) to acquire invasive mesenchymal phenotypes. The EMT-related stemness and chemo-resistance phenotypes are frequently observed in osteotrophic tumors. In this chapter, we will focus on the engagement of the EMT signaling pathway in bone specific homing, quiescence and dormancy phenotypes of the disseminated tumor cells, and the influence of the bone marrow microenvironment in supporting the outgrowth of bone metastatic lesions. We will also discuss the possibility that available therapeutics targeting bone metastasis affect the EMT status of tumor cells.

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.

Breast cancer is a complex, highly heterogenous, and dynamic disease and the leading cause of cancer-related death in women worldwide. Evaluation of the heterogeneity of breast cancer and its various subtypes is crucial to identify novel treatment strategies that can overcome the limitations of currently available options. Explant cultures of human mammary tissue have been known to provide important insights for the study of breast cancer structure and phenotype as they include the context of the surrounding microenvironment, allowing for the comprehensive exploration of patient heterogeneity. However, the major limitation of currently available techniques remains the short-term viability of the tissue owing to loss of structural integrity. Here, an ex vivo culture model using star-shaped poly(ethylene glycol) and maleimide-functionalized heparin (PEG-HM) hydrogels to provide structural support to the explant cultures is presented. The mechanical support allows the culture of the human mammary tissue for up to 3 weeks and prevent disintegration of the cellular structures including the epithelium and surrounding stromal tissue. Further, maintenance of epithelial phenotype and hormonal receptors is observed for up to 2 weeks of culture which makes them relevant for testing therapeutic interventions. Through this study, the importance of donor-to-donor variability and intra-patient tissue heterogeneity is reiterated.

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.

High mammographic density (MD) increases breast cancer (BC) risk and creates a stiff tissue environment. BC risk is also increased in BRCA1/2 gene mutation carriers, which may be in part due to genetic disruption of the tumour suppressor gene Ras association domain family member 1 (RASSF1A), a gene that is also directly regulated by tissue stiffness. High MD combined with BRCA1/2 mutations further increase breast cancer risk, yet BRCA1/2 mutations alone or in combination do not increase MD. The molecular basis for this additive effect therefore remains unclear. We studied the interplay between MD, stiffness, and BRCA1/2 mutation status in human mammary tissue obtained after prophylactic mastectomy from women at risk of developing BC. Our results demonstrate that RASSF1A expression increased in MCF10DCIS.com cell cultures with matrix stiffness up until ranges corresponding with BiRADs 4 stiffnesses (~16 kPa), but decreased in higher stiffnesses approaching malignancy levels (&gt, 50 kPa). Similarly, higher RASSF1A protein was seen in these cells when co-cultivated with high MD tissue in murine biochambers. Conversely, local stiffness, as measured by collagen I versus III abundance, repressed RASSF1A protein expression in BRCA1, but not BRCA2 gene mutated tissues, regional density as measured radiographically repressed RASSF1A in both BRCA1/2 mutated tissues. The combinatory effect of high MD and BRCA mutations on breast cancer risk may be due to RASSF1A gene repression in regions of increased tissue stiffness.

Cold atmospheric plasma (CAP) has emerged as a highly selective anticancer agent, most recently in the form of plasma-activated medium (PAM). Since epithelial–mesenchymal transition (EMT) has been implicated in resistance to various cancer therapies, we assessed whether EMT status is associated with PAM response. Mesenchymal breast cancer cell lines, as well as the mesenchymal variant in an isogenic EMT/MET human breast cancer cell system (PMC42-ET/LA), were more sensitive to PAM treatment than their epithelial counterparts, contrary to their responses to other therapies. The same trend was seen in luminal muscle-invasive bladder cancer model (TSU-Pr1/B1/B2) and the non-muscle-invasive basal 5637 bladder cancer cell line. Three-dimensional spheroid cultures of the bladder cancer cell lines were less sensitive to the PAM treatment compared to their two-dimensional counterparts, however, incrementally better responses were again seen in more mesenchymally-shifted cell lines. This study provides evidence that PAM preferentially inhibits mesenchymally-shifted carcinoma cells, which have been associated with resistance to other therapies. Thus, PAM may represent a novel treatment that can selectively inhibit triple-negative breast cancers and a subset of aggressive bladder cancers, which tend to be more mesenchymal. Our approach may potentially be utilized for other aggressive cancers exhibiting EMT and opens new opportunities for CAP and PAM as a promising new onco-therapy.

Oncogenesis and cancer can arise as a consequence of a wide range of genomic aberrations including mutations, copy number alterations, expression changes and epigenetic modifications encompassing multiple omics layers. Integrating genomic, transcriptomic, proteomic and epigenomic datasets via multi-omics analysis provides the opportunity to derive a deeper and holistic understanding of the development and progression of cancer. There are two primary approaches to integrating multi-omics data: multi-staged (focused on identifying genes driving cancer) and meta-dimensional (focused on establishing clinically relevant tumour or sample classifications). A number of ready-to-use bioinformatics tools are available to perform both multi-staged and meta-dimensional integration of multi-omics data. In this study, we compared nine different integration tools using real and simulated cancer datasets. The performance of the multi-staged integration tools were assessed at the gene, function and pathway levels, while meta-dimensional integration tools were assessed based on the sample classification performance. Additionally, we discuss the influence of factors such as data representation, sample size, signal and noise on multi-omics data integration. Our results provide current and much needed guidance regarding selection and use of the most appropriate and best performing multi-omics integration tools.