Label-free multimodal electro-thermo-mechanical (ETM) phenotyping as a novel biomarker to differentiate between normal, benign, and cancerous breast biopsy tissues.

BACKGROUND: Technologies for quick and label-free diagnosis of malignancies from breast tissues have the potential to be a significant adjunct to routine diagnostics. The biophysical phenotypes of breast tissues, such as its electrical, thermal, and mechanical properties (ETM), have the potential to serve as novel markers to differentiate between normal, benign, and malignant tissue. RESULTS: We report a system-of-biochips (SoB) integrated into a semi-automated mechatronic system that can characterize breast biopsy tissues using electro-thermo-mechanical sensing. The SoB, fabricated on silicon using microfabrication techniques, can measure the electrical impedance (Z), thermal conductivity (K), mechanical stiffness (k), and viscoelastic stress relaxation (%R) of the samples. The key sensing elements of the biochips include interdigitated electrodes, resistance temperature detectors, microheaters, and a micromachined diaphragm with piezoresistive bridges. Multi-modal ETM measurements performed on formalin-fixed tumour and adjacent normal breast biopsy samples from N = 14 subjects were able to differentiate between invasive ductal carcinoma (malignant), fibroadenoma (benign), and adjacent normal (healthy) tissues with a root mean square error of 0.2419 using a Gaussian process classifier. Carcinoma tissues were observed to have the highest mean impedance (110018.8 ± 20293.8 Ω) and stiffness (0.076 ± 0.009 kNm-1) and the lowest thermal conductivity (0.189 ± 0.019 Wm-1 K-1) amongst the three groups, while the fibroadenoma samples had the highest percentage relaxation in normalized load (47.8 ± 5.12%). CONCLUSIONS: The work presents a novel strategy to characterize the multi-modal biophysical phenotype of breast biopsy tissues to aid in cancer diagnosis from small-sized tumour samples. The methodology envisions to supplement the existing technology gap in the analysis of breast tissue samples in the pathology laboratories to aid the diagnostic workflow.

Inhibition of protein translation under matrix-deprivation stress in breast cancer cells.

Matrix-deprivation stress leads to cell-death by anoikis, whereas overcoming anoikis is critical for cancer metastasis. Work from our lab and others has identified a crucial role for the cellular energy sensor AMPK in anoikis-resistance, highlighting a key role for metabolic reprogramming in stress survival. Protein synthesis is a major energy-consuming process that is tightly regulated under stress. Although an increase in protein synthesis in AMPK-depleted experimentally-transformed MEFs has been associated with anoikis, the status and regulation of protein translation in epithelial-origin cancer cells facing matrix-detachment remains largely unknown. Our study shows that protein translation is mechanistically abrogated at both initiation and elongation stages by the activation of the unfolded protein response (UPR) pathway and inactivation of elongation factor eEF2, respectively. Additionally, we show inhibition of the mTORC1 pathway known for regulation of canonical protein synthesis. We further functionally assay this inhibition using SUnSET assay, which demonstrates repression of global protein synthesis in MDA-MB-231 and MCF7 breast cancer cells when subjected to matrix-deprivation. In order to gauge the translational status of matrix-deprived cancer cells, we undertook polysome profiling. Our data revealed reduced but continuous mRNA translation under matrix-deprivation stress. An integrated analysis of transcriptomic and proteomic data further identifies novel targets that may aid cellular adaptations to matrix-deprivation stress and can be explored for therapeutic intervention.

Standardized ready-to-use laboratory protocol to isolate primary human wound associated fibroblasts from infected wound samples for clinical applications.

Owing to the importance of fibroblasts in healing of wounds, it is necessary to isolate and culture them under in vitro conditions for the purpose of understanding the wound biology, drug discovery and development of personalized treatment. Although, several fibroblast cell lines are commercially available, they fail to represent the patient associated parameters. However, establishing a primary fibroblast culture, especially from infected wound samples, is challenging as the sample is more prone to contamination and number of live cells will be minimum in heterogeneous population. Also, it takes lot of efforts and resources for optimization of the protocol to get good quality cell lines from wound samples necessitating multiple trials, resulting in large number of clinical samples to be processed. To the best of our knowledge, for the first time we are reporting the standardized protocol to isolate primary human fibroblasts from acute and chronic wound samples. In this study, various parameters such as explant size (1-2 mm), explant drying time (2 min), transportation and growth culture media (antibiotics (working concentrations 1-3) and serum concentration (10%)) have been optimised. This can be altered for specific needs of cell in terms of both quality and quantity. Outcome of the work provides a ready-to-use protocol, which is very useful to those who want to initiate primary fibroblasts cell culture from infected wound samples either for clinical or research purpose. Further, these cultured primary wound associated fibroblasts have various clinical and biomedical applications in tissue grafting, treatment of burns and scars and wound regeneration especially in non-healing chronic wounds.

Surface-Coated Cerium Nanoparticles to Improve Chemotherapeutic Delivery to Tumor Cells.

The antioxidant property of cerium oxide nanoparticles has increased their demand as a nanocarrier to improve the delivery and therapeutic efficacy of anticancer drugs. Here, we report the synthesis of alginate-coated ceria nanoformulations (ceria NPs) and characterization using FTIR spectroscopy, Raman microscopy, and X-ray diffraction. The synthesized ceria NPs show negligible inherent in vitro toxicity when tested on a MDA-MB-231 breast cancer cell line at higher particle concentrations. Upon loading these particles with doxorubicin (Dox) and paclitaxel (PTX) drugs, we observe a potential synergistic cytotoxic effect mediated by the drug and the ceria NPs, resulting in the better killing capacity as well as suppression of cell migration against the MDA-MB-231 cell line. Further, to verify the immune-escaping capacity before targeting cancer cells, we coated the drug-loaded ceria NPs with the membrane of MDA-MB-231 cells using an extrusion method. The resultant delivery system exhibited in vitro preferential uptake by the MDA-MB-231 cell line and showed reduced uptake by the murine macrophage cell line (RAW 264.7), assigning its potential application as non-immunogenic personalized therapy in targeting and killing of cancer cells.

Human primary chronic wound derived fibroblasts demonstrate differential pattern in expression of fibroblast specific markers, cell cycle arrest and reduced proliferation.

INTRODUCTION: Although wound refers to simple cut in the skin, most wounds don't heal because of the various local and systemic factors that lead to its complexity and chronicity. Thus, prior understanding of the status of the wound is necessary and methods that can differentiate between the healing and non-healing wounds at a much earlier stage is crucial for a successful treatment. METHODS: The current study aims at differentiating Acute Wound Fibroblasts (AWFs) and Chronic Wound Fibroblasts (CWFs) based on differential expression of fibroblast specific markers such as Vimentin and Alpha Smooth Muscle Actin (α-SMA) and compare its cell cycle and proliferation. RESULTS: Immunostaining and western blotting analysis showed that, AWFs and CWFs differentially expressed vimentin and α-SMA, with AWFs and CWFs showing higher expression of vimentin and α-SMA respectively. AWFs showed higher distributions in G0/G1 (67.43% vs. 62.16%), S phase (22.61% vs. 8.51%) compared to CWFs. However, AWFs showed decreased distributions compared to CWFs in G2 + M phase (8.14% vs. 10.6%). Thus, it was observed that CWFs showed cell cycle arrest in the G1/G0 phase and inhibited DNA synthesis, which was further confirmed by reduced proliferation of CWFs. We suggest that, differential expression of the cell specific markers can be attributed to its pathophysiological status and chronicity of the wound and reduced proliferation rate of CWFs is due to lesser expression of vimentin, which is a key protein for in vitro cell proliferation. CONCLUSIONS: Outcome of the study serve as an immunological tool to guide the chronicity of the wound, which helps to understand the wound towards design of personalized care. The findings also represent a promising opportunity to gain insight into how cell cycle arrest can impact on wound healing and clinical outcomes.

AMP-activated protein kinase promotes breast cancer stemness and drug resistance.

Breast cancer stem cells (BCSCs) are a major cause of therapy resistance and tumour progression. Currently, their regulation is not entirely understood. Previous work from our laboratory demonstrated a context-specific pro-tumorigenic role for AMP-activated protein kinase (AMPK) under anchorage-deprivation and mammosphere formation, which are hallmarks of BCSCs. Therefore, we investigated the role of AMPK in the maintenance of BCSC state/function. AMPK depletion reduces serial sphere formation in vitro and tumour initiation in vivo. Intriguingly, tumour-derived cell analysis using stem cell markers and functional assays revealed that AMPK is required for the maintenance of BCSC populations in vivo. AMPK promotes the expression of stemness genes such as NANOG, SOX2 and BMI1 through the transcriptional upregulation of TWIST via promoter acetylation. Further, AMPK-driven stemness plays a critical role in doxorubicin resistance. Significantly, AMPK activity increased after chemotherapy in patient-derived tumour samples alongside an increase in stemness markers. Importantly, AMPK depletion sensitises mouse tumours to doxorubicin treatment. Our work indicates that targeting of AMPK in conjunction with regular chemotherapy is likely to reduce the stem cell pool and improve chemosensitivity in breast cancers.

Somatic mutation analyses of stem-like cells in gingivobuccal oral squamous cell carcinoma reveals DNA damage response genes.

Gingivobuccal oral squamous cell carcinoma (OSCC-GB) occurs among persons who excessively chew smokeless tobacco in India. To understand the role of cancer stem cells (CSCs) in the disease, we have performed transcriptomics analysis on RNA-seq data from OSCC-GB primary tumors. The mutational signature analysis of the identified novel and Catalogue of Somatic Mutations in Cancer (COSMIC) variants reveals DNA damage associated etiology based on identified COSMIC signatures showing a higher prevalence of C > T mutations and 1 bp T/(A) nucleotide insertions, pointing to the role of smokeless tobacco carcinogens. The differential somatic mutational, functional impact predictions, and survival analysis reveals the role of DNA damage response-related genes, with the CREBBP gene as a major player. The new CSC somatic variants identified in the study may play a crucial role in cancer metastasis, local-regional recurrence, chemo- and/or radioresistance that contributes to high mortality of the Indian OSCC-GB patients.

PD-1 and PD-L1 Expression in Indian Women with Breast Cancer.

OBJECTIVE: The interaction between programmed cell death protein 1 (PD-1) on activated T-lymphocytes and programmed death-ligand 1 (PD-L1) on tumor cells or antigen-presenting cells sends immunosuppressive signals leading to the escape of tumor cells from the host anti-tumor immune response. Inhibiting this interaction with antibodies against PD-1 or PD-L1 is emerging as a valuable therapeutic strategy. However, tissue distribution patterns for PD-L1 and PD-1 in breast cancer patients from India are not reported, yet many clinical trials are underway. In this study the expression of PD-1 and PD-L1 in breast cancer patient samples from India was characterized. MATERIALS AND METHODS: The study included 392 cases of operated breast cancer (2012-2017) from a tertiary cancer care center in Bangalore, Karnataka, India. Paraffin blocks were retrievable and receptor status was known. Immunohistochemistry (IHC) was performed using anti-PD-L1 and anti-PD-1 antibodies. RNA was isolated from 76 fresh tumors and nine adjacent normal tissues (2019). PD-L1 transcript levels were measured by RT-qPCR using Hypoxanthine-guanine phosphoribosyl transferase (HPRT) as a reference gene. RESULTS: Based on IHC, PD-1 expression within tumor-infiltrating immune cells (TIICs) was observed in 55/385 cases (14%) across all breast cancer types. In triple-negative breast cancer (TNBC), 21/132 cases (16%) showed PD-1 staining in TIICs. The overall expression of PD-L1 in breast tumor cells across all breast cancer subtypes and TIICs was 11% (41/378) and 39% (151/385), respectively. A relatively higher proportion of TNBC cases had PD-L1 expression in tumor cells (17/132 cases, 13%) and immune cells (68/132 cases, 52%). We also detected PD-L1 transcript expression by qRT-PCR in freshly isolated tumor samples. CONCLUSION: These findings show that around 52% (68/132) of the TNBC cases express PD-L1 in TIICs. Hence, anti-PD-1/PD-L1 therapy alone or combined with chemotherapy may be a promising treatment for TNBC in Indian patients.

RapidET: a MEMS-based platform for label-free and rapid demarcation of tumors from normal breast biopsy tissues.

The rapid and label-free diagnosis of malignancies in ex vivo breast biopsy tissues has significant utility in pathology laboratories and operating rooms. We report a MEMS-based platform integrated with microchips that performs phenotyping of breast biopsy tissues using electrothermal sensing. The microchip, fabricated on a silicon substrate, incorporates a platinum microheater, interdigitated electrodes (IDEs), and resistance temperature detectors (RTDs) as on-chip sensing elements. The microchips are integrated onto the platform using a slide-fit contact enabling quick replacement for biological measurements. The bulk resistivity (ρ B ), surface resistivity (ρ S ), and thermal conductivity (k) of deparaffinized and formalin-fixed paired tumor and adjacent normal breast biopsy samples from N = 8 patients were measured. For formalin-fixed samples, the mean ρ B for tumors showed a statistically significant fold change of 4.42 (P = 0.014) when the tissue was heated from 25 °C to 37 °C compared to the adjacent normal tissue, which showed a fold change of 3.47. The mean ρ S measurements also showed a similar trend. The mean k of the formalin-fixed tumor tissues was 0.309 ± 0.02 W m-1 K-1 compared to a significantly higher k of 0.563 ± 0.028 W m-1 K-1 for the adjacent normal tissues. A similar trend was observed in ρ B, ρ S, and k for the deparaffinized tissue samples. An analysis of a combination of ρ B , ρ S , and k using Fisher's combined probability test and linear regression suggests the advantage of using all three parameters simultaneously for distinguishing tumors from adjacent normal tissues with higher statistical significance.

Sustained AMPK Activation and Proline Metabolism Play Critical Roles in the Survival of Matrix-Deprived Transformed Cells.

Attachment to the matrix is critical for the survival of adherent cells, whereas detachment triggers death by apoptosis. Therefore, solid tumors must acquire the ability to survive the stress of matrix-detachment to transit through circulation and seed metastases. Although a central role for energy metabolism in cancer progression is well established, what distinguishes its role in the cellular state of the matrix-deprived form compared to the matrix-attached form is not fully understood yet. Using an in vitro transformation model dependent on simian virus 40 (SV40) small t (ST) antigen for cellular survival and proliferation in matrix-deprived conditions, we demonstrate that 5'-adenosine monophosphate-activated protein kinase (AMPK) activity is elevated and sustained under matrix-deprived conditions in ST-expressing fibroblasts. Additionally, these cells display elevated energy (ATP) levels under matrix-deprived conditions in contrast to cells lacking ST expression. The elevated ATP levels are coupled to increased levels of proline in ST-expressing cells, as revealed by metabolomics studies. The AMPK-dependent upregulation of proline oxidase, an enzyme of proline degradation, is a key link for elevated ATP levels. This functional link is further established by proline supplementation concomitant with AMPK activation in matrix-deprived cells lacking ST antigen, yielding ATP and enhancing survival. Thus, our data establishes a key role for AMPK-dependent regulation of proline metabolism in mediating energy homeostasis and promoting survival of matrix-deprived cells. These findings identify key markers that distinguish the metabolic states of matrix-detached and matrix-attached transformed cells and have implications in developing novel therapeutic strategies for specifically targeting matrix-detached metastasizing cancer cells.

Correction: Targeting focal adhesion kinase overcomes erlotinib resistance in smoke induced lung cancer by altering phosphorylation of epidermal growth factor receptor.

[This corrects the article DOI: 10.18632/oncoscience.395.].

3D Tumor Models for Breast Cancer: Whither We Are and What We Need.

Three-dimensional (3D) models have led to a paradigm shift in disease modeling in vitro, particularly for cancer. The past decade has seen a phenomenal increase in the development of 3D models for various types of cancers with a focus on studying stemness, invasive behavior, angiogenesis, and chemoresistance of cancer cells, as well as contributions of its stroma, which has expanded our understanding of these processes. Cancer biology is moving into exploring the emerging hallmarks of cancer, such as inflammation, immune evasion, and reprogramming of energy metabolism. Studies into these emerging concepts have provided novel targets and treatment options such as antitumor immunotherapy. However, 3D models that can investigate the emerging hallmarks are few and underexplored. As commonly used immunocompromised mice and syngenic mice cannot accurately mimic human immunology, stromal interactions, and metabolism and require the use of prohibitively expensive humanized mice, there is tremendous scope to develop authentic 3D tumor models in these areas. Taking the specific case of breast cancer, we discuss the currently available 3D models, their applications to mimic signaling in cancer, tumor-stroma interactions, drug responses, and assessment of drug delivery systems and therapies. We discuss the lacunae in the development of 3D tumor models for the emerging hallmarks of cancer, for lesser-explored forms of breast cancer, and provide insights to develop such models. We discuss how the next generation of 3D models can provide a better mimic of human cancer modeling compared to xenograft models and the scope toward preclinical models and precision medicine.

Myofibroblast progeny in wound biology and wound healing studies.

Fibroblasts and myofibroblasts play a myriad of important roles in human tissue function, especially in wound repair and healing. Among all cells, fibroblasts are group of cells that decide the status of wound as they maintain tissue homeostasis. Currently, the increase in the deleterious effects of chronic wound and their morbidity rate has necessitated the need to understand the influence of fibroblasts and myofibroblasts, which chiefly originate locally from tissue-resident fibroblasts to address the same. Wound pathophysiology is complex, herein, we have discussed fibroblast and myofibroblast heterogeneity in skin and different organs by understanding the phenotypical and functional properties of each of its sub-populations in the process of wound healing. Recent advancements in fibroblast activation, differentiation to myofibroblasts, proliferation and migration are discussed in detail. Fibroblasts and myofibroblasts are key players in wound healing and wound remodelling, respectively, and their significance in wound repair is discussed. An increased understanding of their biology during wound healing also gives an opportunity to explore more of fibroblast and myofibroblast focused therapies to treat chronic wounds which are clinical challenges. In this regard, in the current review, we have described the different methods for isolation of primary fibroblasts and myofibroblasts from both animal models and humans, and their characterization. Additionally, we have also provided details on possible molecular targets for better understanding of prognosis, diagnosis and treatment of chronic wounds. Information will help both researchers and clinicians in providing molecular insight that enable them for effective chronic wound management. The knowledge of intimate dialogue between the fibroblast, sub-populations like, myofibroblast and their microenvironment, will serve useful in determining novel, efficient and specific therapeutic targets to treat pathological wound conditions.

Multi-Stability and Consequent Phenotypic Plasticity in AMPK-Akt Double Negative Feedback Loop in Cancer Cells.

Adaptation and survival of cancer cells to various stress and growth factor conditions is crucial for successful metastasis. A double-negative feedback loop between two serine/threonine kinases AMPK (AMP-activated protein kinase) and Akt can regulate the adaptation of breast cancer cells to matrix-deprivation stress. This feedback loop can significantly generate two phenotypes or cell states: matrix detachment-triggered pAMPKhigh/ pAktlow state, and matrix (re)attachment-triggered pAkthigh/ pAMPKlow state. However, whether these two cell states can exhibit phenotypic plasticity and heterogeneity in a given cell population, i.e., whether they can co-exist and undergo spontaneous switching to generate the other subpopulation, remains unclear. Here, we develop a mechanism-based mathematical model that captures the set of experimentally reported interactions among AMPK and Akt. Our simulations suggest that the AMPK-Akt feedback loop can give rise to two co-existing phenotypes (pAkthigh/ pAMPKlow and pAMPKhigh/pAktlow) in specific parameter regimes. Next, to test the model predictions, we segregated these two subpopulations in MDA-MB-231 cells and observed that each of them was capable of switching to another in adherent conditions. Finally, the predicted trends are supported by clinical data analysis of The Cancer Genome Atlas (TCGA) breast cancer and pan-cancer cohorts that revealed negatively correlated pAMPK and pAkt protein levels. Overall, our integrated computational-experimental approach unravels that AMPK-Akt feedback loop can generate multi-stability and drive phenotypic switching and heterogeneity in a cancer cell population.

Challenges in Healing Wound: Role of Complementary and Alternative Medicine.

Although the word wound sounds like a simple injury to tissue, individual's health status and other inherent factors may make it very complicated. Hence, wound healing has gained major attention in the healthcare. The biology wound healing is precise and highly programmed, through phases of hemostasis, inflammation, proliferation and remodeling. Current options for wound healing which includes, use of anti-microbial agents, healing promoters along with application of herbal and natural products. However, there is no efficient evidence-based therapy available for specific chronic wounds that can result in definitive clinical outcomes. Under co-morbid conditions, chronic would poses numerous challenges. Use of Complementary and Alternative Medicines (CAMs) in health care sector is increasing and its applications in wound management remains like to "separate the diamonds from ore." Attempts have been made to understand the wound at the molecular level, mainly through the analysis of signature genes and the influence of several synthetic and natural molecules on these. We have outlined a review of challenges in chronic wound healing and the role of CAMs in chronic wound management. The main focus is on the applications and limitations of currently available treatment options for a non-healing wound and the best possible alternates to consider. This information generates broader knowledge on challenges in chronic wound healing, which can be further addressed using multidisciplinary approach and combination therapies.

Magnetic nanofibers based bandage for skin cancer treatment: a non-invasive hyperthermia therapy.

BACKGROUND: The treatment of non-melanoma skin cancer and deadliest malignant melanoma skin cancer are the fifth and ninth most expensive treatments in Medicare, respectively. Moreover, the recurrence of cancer after currently available therapies, that is, surgery or radiotherapy, reduces the patient's life expectancy. AIMS: In view of this, we fabricated magnetic nanofibrous mat-based bandage to treat skin cancer non-invasively using an external alternating current (AC) magnetic field induced hyperthermia. METHODS: The Fe3 O4 nanoparticles incorporated polycaprolactone (PCL) fibers based bandages were fabricated using the electrospinning technique. The efficacy of the bandage was investigated in vitro using parental/doxorubicin hydrochloride (Dox)-resistant HeLa cells and in vivo using BALB/c mouse model in the presence of an external AC magnetic field (AMF). RESULTS: The PCL-Fe3 O4 fibrous mat-based bandages dissipate heat energy locally on the application of an external AMF and increase the surrounding temperature in a controlled way up to 45°C in a few mins. The in vitro study confirms the elevated temperature could kill parental and Dox-resistant HeLa cells significantly. As the activity of Dox enhanced at a higher temperatures, more than 85% of parental HeLa cells were dead when cells incubated with Dox contained fibrous mat in the presence of AMF for 10 minutes. Further, we confirm the full recovery of chemically induced skin tumors on BALB/c mice within a month after five hyperthermic doses for 15 minutes. Also, there was no sign of inflammation and recurrence of cancer post-therapy. CONCLUSION: The present study confirms the PCL-Fe3 O4 nanofibrous based bandages are unique and compelling to treat skin cancer.

Epigenetic feedback and stochastic partitioning during cell division can drive resistance to EMT.

Epithelial-mesenchymal transition (EMT) and its reverse process mesenchymal-epithelial transition (MET) are central to metastatic aggressiveness and therapy resistance in solid tumors. While molecular determinants of both processes have been extensively characterized, the heterogeneity in the response of tumor cells to EMT and MET inducers has come into focus recently, and has been implicated in the failure of anti-cancer therapies. Recent experimental studies have shown that some cells can undergo an irreversible EMT depending on the duration of exposure to EMT-inducing signals. While the irreversibility of MET, or equivalently, resistance to EMT, has not been studied in as much detail, evidence supporting such behavior is slowly emerging. Here, we identify two possible mechanisms that can underlie resistance of cells to undergo EMT: epigenetic feedback in ZEB1/GRHL2 feedback loop and stochastic partitioning of biomolecules during cell division. Identifying the ZEB1/GRHL2 axis as a key determinant of epithelial-mesenchymal plasticity across many cancer types, we use mechanistic mathematical models to show how GRHL2 can be involved in both the abovementioned processes, thus driving an irreversible MET. Our study highlights how an isogenic population may contain subpopulation with varying degrees of susceptibility or resistance to EMT, and proposes a next set of questions for detailed experimental studies characterizing the irreversibility of MET/resistance to EMT.

A Novel Ex Vivo System Using 3D Polymer Scaffold to Culture Circulating Tumor Cells from Breast Cancer Patients Exhibits Dynamic E-M Phenotypes.

The majority of the cancer-associated deaths is due to metastasis-the spread of tumors to other organs. Circulating tumor cells (CTCs), which are shed from the primary tumor into the circulation, serve as precursors of metastasis. CTCs have now gained much attention as a new prognostic and diagnostic marker, as well as a screening tool for patients with metastatic disease. However, very little is known about the biology of CTCs in cancer metastasis. An increased understanding of CTC biology, their heterogeneity, and interaction with other cells can help towards a better understanding of the metastatic process, as well as identify novel drug targets. Here we present a novel ex vivo 3D system for culturing CTCs from breast cancer patient blood samples using porous poly(ε-caprolactone) (PCL) scaffolds. As a proof of principle study, we show that ex vivo culture of 12/16 (75%) advanced stage breast cancer patient blood samples were enriched for CTCs identified as CK+ (cytokeratin positive) and CD45- (CD45 negative) cells. The deposition of extracellular matrix proteins on the PCL scaffolds permitted cellular attachment to these scaffolds. Detection of Ki-67 and bromodeoxyuridine (BrdU) positive cells revealed proliferating cell population in the 3D scaffolds. The CTCs cultured without prior enrichment exhibited dynamic differences in epithelial (E) and mesenchymal (M) composition. Thus, our 3D PCL scaffold system offers a physiologically relevant model to be used for studying CTC biology as well as for individualized testing of drug susceptibility. Further studies are warranted for longitudinal monitoring of epithelial-mesenchymal transition (EMT) in CTCs for clinical association.

A Composite of Hyaluronic Acid-Modified Graphene Oxide and Iron Oxide Nanoparticles for Targeted Drug Delivery and Magnetothermal Therapy.

Graphene oxide (GO) nanoparticles have been developed for a variety of biomedical applications as a number of different therapeutic modalities may be added onto them. Here, we report the development and testing of such a multifunctional GO nanoparticle platform that contains a grafted cell-targeting functionality, active pharmaceutical ingredients, and particulates that enable the use of magnetothermal therapy. Specifically, we demonstrate the ability to covalently attach hyaluronic acid (HA) onto GO, and the resultant nanoparticulates (GO-HA) exhibited low inherent toxicity toward two different breast cancer cell lines, BT-474 and MDA-MB-231. Doxorubicin (Dox) and paclitaxel (Ptx) were successfully loaded onto GO-HA with high and moderate efficiencies, respectively. A GO-HA-Dox/Ptx system was significantly better than the GO-Dox/Ptx system at specifically killing CD44-expressing MDA-MB-231 cells but not BT-474 cells that do not express CD44. Further, modified iron oxide nanoparticles were loaded onto the GO-HA-Dox system, enabling the use of magnetic hyperthermia. Hyperthermia in combination with Dox treatment through the GO-HA system showed significantly better performance in reducing viable tumor cell numbers when compared to the individual systems. In summary, we showcase a multifunctional GO nanoparticle system that demonstrates improved efficacy in killing tumor cells.