Multicellular Tumoroids for Investigating Cancer Stem-Like Cells in the Heterogeneous Tumor Microenvironment.

Cancer stem-like cells (CSC) are a major contributing factor to chemoresistance, tumor recurrence, and poor survival outcomes in patients across cancer types. Signaling from non-tumor cells in the tumor microenvironment (TME) enriches for and supports CSC. This complex cell-cell signaling in the heterogeneous TME presents a challenge for patient survival; however, it also presents an opportunity to develop new targeted therapies that can inhibit survival of CSC. In this chapter, we report a multicellular tumoroid model which can be used to investigate the interactions between cancer cells and non-tumor cells in the TME to better understand the contribution of various cell types to cancer cell phenotypes, as well as the underlying mechanisms involved. The following methods allow for each cell type to be distinguished using FACS and studied individually. Gene expression can be analyzed for cancer cells, as well as the other non-tumor cells using qPCR following sorting. The response to chemotherapeutic agents and expression of stem markers can be determined for cancer cells using flow cytometry, excluding the other cell types to get an accurate view of the cancer cells. Furthermore, the viability of non-tumor cells can be analyzed as well to determine if there are cytotoxic effects of the drugs on non-tumor cells. Thus, the multicellular tumoroid model will reveal the interactions between the CSC and non-tumor cells in the heterogenous TME, resulting in discoveries in the fields of cancer biology, novel targeted therapies, and personalized drug screening for precision medicine.

Co-Delivery Polymeric Poly(Lactic-Co-Glycolic Acid) (PLGA) Nanoparticles to Target Cancer Stem-Like Cells.

Nanoparticle drug delivery has been promoted as an effective mode of delivering antineoplastic therapeutics. However, most nanoparticle designs fail to consider the multifaceted tumor microenvironment (TME) that produce pro-tumoral niches, which are often resistant to chemo- and targeted therapies. In order to target the chemoresistant cancer stem-like cells (CSCs) and their supportive TME, in this chapter we describe a nanoparticle-based targeted co-delivery that addresses the paracrine interactions between CSC and non-cancerous mesenchymal stem cells (MSCs) in the TME. Carcinoma-activated MSCs have been shown to increase the chemoresistance and metastasis of CSC. Yet their contributions to protect the CSC TME have not yet been systematically investigated in the design of nanoparticles for drug delivery. Therefore, we describe the fabrication of degradable poly(lactic-co-glycolic acid) (PLGA) nanoparticles (120-200 nm), generated with an electrospraying process that encapsulates both a conventional chemotherapeutic, paclitaxel, and a targeted tyrosine kinase inhibitor, sunitinib, to limit MSC interactions with CSC. In the 3D hetero-spheroid model that comprises both CSCs and MSCs, the delivery of sunitinib as a free drug disrupted the MSC-protected CSC stemness and migration. Therefore, this chapter describes the co-delivery of paclitaxel and sunitinib via PLGA nanoparticles as a potential targeted therapy strategy for targeting CSCs. Overall, nanoparticles can provide an effective delivery platform for targeting CSCs and their TME together. Forthcoming studies can corroborate similar combined therapies with nanoparticles to improve the killing of CSC and chemoresistant cancer cells, thereby improving treatment efficiency.

Applications of Innovation Technologies for Personalized Cancer Medicine: Stem Cells and Gene-Editing Tools.

Personalized medicine is a new approach toward safer and even cheaper treatments with minimal side effects and toxicity. Planning a therapy based on individual properties causes an effective result in a patient's treatment, especially in a complex disease such as cancer. The benefits of personalized medicine include not only early diagnosis with high accuracy but also a more appropriate and effective therapeutic approach based on the unique clinical, genetic, and epigenetic features and biomarker profiles of a specific patient's disease. In order to achieve personalized cancer therapy, understanding cancer biology plays an important role. One of the crucial applications of personalized medicine that has gained consideration more recently due to its capability in developing disease therapy is related to the field of stem cells. We review various applications of pluripotent, somatic, and cancer stem cells in personalized medicine, including targeted cancer therapy, cancer modeling, diagnostics, and drug screening. CRISPR-Cas gene-editing technology is then discussed as a state-of-the-art biotechnological advance with substantial impacts on medical and therapeutic applications. As part of this section, the role of CRISPR-Cas genome editing in recent cancer studies is reviewed as a further example of personalized medicine application.

Gender bias in reference letters for residency and academic medicine: a systematic review.

Reference letters play an important role for both postgraduate residency applications and medical faculty hiring processes. This study seeks to characterise the ways in which gender bias may manifest in the language of reference letters in academic medicine. In particular, we conducted a systematic review in accordance with Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. We searched Embase, MEDLINE and PsycINFO from database inception to July 2020 for original studies that assessed gendered language in medical reference letters for residency applications and medical faculty hiring. A total of 16 studies, involving 12 738 letters of recommendation written for 7074 applicants, were included. A total of 32% of applicants were women. There were significant differences in how women were described in reference letters. A total of 64% (7/11) studies found a significant difference in gendered adjectives between men and women. Among the 7 studies, a total of 86% (6/7) noted that women applicants were more likely to be described using communal adjectives, such as "delightful" or "compassionate", while men applicants were more likely to be described using agentic adjectives, such as "leader" or "exceptional". Several studies noted that reference letters for women applicants had more frequent use of doubt raisers and mentions of applicant personal life and/or physical appearance. Only one study assessed the outcome of gendered language on application success, noting a higher residency match rate for men applicants. Reference letters within medicine and medical education exhibit language discrepancies between men and women applicants, which may contribute to gender bias against women in medicine.

Surfaces with instant and persistent antimicrobial efficacy against bacteria and SARS-CoV-2.

Surfaces contaminated with bacteria and viruses contribute to the transmission of infectious diseases and pose a significant threat to global public health. Modern day disinfection either relies on fast-acting (>3-log reduction within a few minutes), yet impermanent, liquid-, vapor-, or radiation-based disinfection techniques, or long-lasting, but slower-acting, passive antimicrobial surfaces based on heavy metal surfaces, or metallic nanoparticles. There is currently no surface that provides instant and persistent antimicrobial efficacy against a broad spectrum of bacteria and viruses. In this work, we describe a class of extremely durable antimicrobial surfaces incorporating different plant secondary metabolites that are capable of rapid disinfection (>4-log reduction) of current and emerging pathogens within minutes, while maintaining persistent efficacy over several months and under significant environmental duress. We also show that these surfaces can be readily applied onto a variety of desired substrates or devices via simple application techniques such as spray, flow, or brush coating.

Atovaquone: An Inhibitor of Oxidative Phosphorylation as Studied in Gynecologic Cancers.

Oxidative phosphorylation is an active metabolic pathway in cancer. Atovaquone is an oral medication that inhibits oxidative phosphorylation and is FDA-approved for the treatment of malaria. We investigated its potential anti-cancer properties by measuring cell proliferation in 2D culture. The clinical formulation of atovaquone, Mepron, was given to mice with ovarian cancers to monitor its effects on tumor and ascites. Patient-derived cancer stem-like cells and spheroids implanted in NSG mice were treated with atovaquone. Atovaquone inhibited the proliferation of cancer cells and ovarian cancer growth in vitro and in vivo. The effect of atovaquone on oxygen radicals was determined using flow and imaging cytometry. The oxygen consumption rate (OCR) in adherent cells was measured using a Seahorse XFe96 Extracellular Flux Analyzer. Oxygen consumption and ATP production were inhibited by atovaquone. Imaging cytometry indicated that the majority of the oxygen radical flux triggered by atovaquone occurred in the mitochondria. Atovaquone decreased the viability of patient-derived cancer stem-like cells and spheroids implanted in NSG mice. NMR metabolomics showed shifts in glycolysis, citric acid cycle, electron transport chain, phosphotransfer, and metabolism following atovaquone treatment. Our studies provide the mechanistic understanding and preclinical data to support the further investigation of atovaquone's potential as a gynecologic cancer therapeutic.

Injectable three-dimensional tumor microenvironments to study mechanobiology in ovarian cancer.

Epithelial ovarian cancers are among the most aggressive forms of gynecological malignancies. Despite the advent of poly adenosine diphosphate-ribose polymerase (PARP) and checkpoint inhibitors, improvement to patient survival has been modest. Limited in part by clinical translation, beneficial therapeutic strategies remain elusive in ovarian cancers. Although elevated levels of extracellular proteins, including collagens, proteoglycans, and glycoproteins, have been linked to chemoresistance, they are often missing from the processes of drug- development and screening. Biophysical and biochemical signaling from the extracellular matrix (ECM) determine cellular phenotype and affect both tumor progression and therapeutic response. However, many state-of-the-art tumor models fail to mimic the complexities of the tumor microenvironment (TME) and omit key signaling components. In this article, two interpenetrating network (IPN) hydrogel scaffold platforms, comprising of alginate-collagen or agarose-collagen, have been characterized for use as 3D in vitro models of epithelial ovarian cancer ECM. These highly tunable, injection mold compatible, and inexpensive IPNs replicate the critical governing physical and chemical signaling present within the ovarian TME. Additionally, an effective and cell-friendly live-cell retrieval method has been established to recover cells post-encapsulation. Lastly, functional mechanotransduction in ovarian cancers was demonstrated by increasing scaffold stiffness within the 3D in vitro ECM models. With these features, the agarose-collagen and alginate-collagen hydrogels provide a robust TME for the study of mechanobiology in epithelial cancers. STATEMENT OF SIGNIFICANCE: Ovarian cancer is the most lethal gynecologic cancer afflicting women today. Here we present the development, characterization, and validation of 3D interpenetrating platforms to shift the paradigm in standard in vitro modeling. These models help elucidate the roles of biophysical and biochemical cues in ovarian cancer progression. The agarose-collagen and alginate-collagen interpenetrating network (IPN) hydrogels are simple to fabricate, inexpensive, and can be modified to create custom mechanical stiffnesses and concentrations of bio-adhesive motifs. Given that investigations into the roles of biophysical characteristics in ovarian cancers have provided incongruent results, we believe that the IPN platforms will be critically important to uncovering molecular drivers. We also expect these platforms to be broadly applicable to studies involving mechanobiology in solid tumors.

A Bioreactor for 3D In Vitro Modeling of the Mechanical Stimulation of Osteocytes.

The bone is a mechanosensitive organ that is also a common metastatic site for prostate cancer. However, the mechanism by which the tumor interacts with the bone microenvironment to further promote disease progression remains to be fully understood. This is largely due to a lack of physiological yet user-friendly models that limit our ability to perform in-depth mechanistic studies. Here, we report a tunable bioreactor which facilitates the 3D culture of the osteocyte cell line, MLO-Y4, in a hydroxyapatite/tricalcium phosphate (HA/TCP) scaffold under constant fluidic shear stress and tunable hydrostatic pressure within physiological parameters. Increasing hydrostatic pressure was sufficient to induce a change in the expression of several bone remodeling genes such as Dmp1, Rankl, and Runx2. Furthermore, increased hydrostatic pressure induced the osteocytes to promote the differentiation of the murine macrophage cell line RAW264.7 toward osteoclast-like cells. These results demonstrate that the bioreactor recapitulates the mechanotransduction response of osteocytes to pressure including the measurement of their functional ability in a 3D environment. In conclusion, the bioreactor would be useful for exploring the mechanisms of osteocytes in bone health and disease.

The Role of the Tumor Microenvironment in CSC Enrichment and Chemoresistance: 3D Co-culture Methods.

Cancer stem-like cells (CSC) are responsible for tumor progression, chemoresistance, recurrence, and poor outcomes in many cancers, making them critical research and therapeutic targets. One of the critical components potentiating CSC chemoresistance is the interactions between CSC and the surrounding cells in the tumor microenvironment. Our lab has developed several 3D co-culture models to study ovarian CSC interactions with stromal or immune cells found in ovarian tumor microenvironments. In this chapter, we use ovarian cancer as a model to describe the methodologies developed in our lab; however, these techniques are applicable to a wide range of cancers. First, we discuss our method for isolating CSC from heterogeneous tumors and for creating 3D self-assembled tumoroids in hanging drop plates, in either monoculture or co-culture with mesenchymal stem cells or monocytes/macrophages. We then discuss methods for analyzing these models with a focus on isolating cell-type-specific changes and mechanism investigation. Specifically, we describe lentiviral transduction and flow cytometry as established and robust methods to identify and separate each cell type for downstream analysis. We then describe methods to examine CSC functionality with transwell migration assays and colorimetric MTS-based proliferation assays. Finally, we demonstrate enzyme-linked immunosorbent assays (ELISA ) and quantitative polymerase chain reaction (qPCR) methods as mechanistic investigation tools to decouple paracrine and juxtacrine interactions. These methods have wide-reaching applications in cancer research from basic biological investigations, to drug discovery, and personalized drug screening for precision medicine.

Design and applications of surfaces that control the accretion of matter.

Surfaces that provide control over liquid, solid, or vapor accretion provide an evolutionary advantage to numerous plants, insects, and animals. Synthetic surfaces inspired by these natural surfaces can have a substantial impact on diverse commercial applications. Engineered liquid and solid repellent surfaces are often designed to impart control over a single state of matter, phase, or fouling length scale. However, surfaces used in diverse real-world applications need to effectively control the accrual of matter across multiple phases and fouling length scales. We discuss the surface design strategies aimed at controlling the accretion of different states of matter, particularly those that work across multiple length scales and different foulants. We also highlight notable applications, as well as challenges associated with these designer surfaces' scale-up and commercialization.

Novel Omniphobic Platform for Multicellular Spheroid Generation, Drug Screening, and On-Plate Analysis.

Multicellular spheroids are superior to other culture geometries in reproducing critical physiological conditions of tumors, such as the diffusion of oxygen, nutrients, waste, and drugs, leading to a more precise model of in vivo drug sensitivity and resistance. Previously reported spheroid culture platforms are often difficult to use, expensive, single-use, or mechanically unstable. Here, we report a facile, mechanically stable, high-throughput spheroid culture platform based on hierarchically textured omniphobic surfaces. The developed omniphobic surfaces display very high contact angles with a range of different liquids, including the cell-laden culture media, thereby minimizing the cell surface contact area. Additionally, these surfaces maintain these high contact angles for extended periods of time to ensure cell aggregation. Using this novel platform, we demonstrate the generation and maintenance of robust multicellular spheroids, as well as heterogeneous, multicell-type spheroids. The platform is extremely robust, resistant to mechanical shock, allows for on-plate imaging, and is also the first-ever spheroid generation platform that can be reused repeatedly. Finally, the platform is suitable for on-plate drug screening and enables the first-ever, on-plate immunofluorescence staining and imaging of spheroids.

Personalized models of heterogeneous 3D epithelial tumor microenvironments: Ovarian cancer as a model.

Intractable human diseases such as cancers, are context dependent, unique to both the individual patient and to the specific tumor microenvironment. However, conventional cancer treatments are often nonspecific, targeting global similarities rather than unique drivers. This limits treatment efficacy across heterogeneous patient populations and even at different tumor locations within the same patient. Ultimately, this poor efficacy can lead to adverse clinical outcomes and the development of treatment-resistant relapse. To prevent this and improve outcomes, it is necessary to be selective when choosing a patient's optimal adjuvant treatment. In this review, we posit the use of personalized, tumor-specific models (TSM) as tools to achieve this remarkable feat. First, using ovarian cancer as a model disease, we outline the heterogeneity and complexity of both the cellular and extracellular components in the tumor microenvironment. Then we examine the advantages and disadvantages of contemporary cancer models and the rationale for personalized TSM. We discuss how to generate precision 3D models through careful and detailed analysis of patient biopsies. Finally, we provide clinically relevant applications of these versatile personalized cancer models to highlight their potential impact. These models are ideal for a myriad of fundamental cancer biology and translational studies. Importantly, these approaches can be extended to other carcinomas, facilitating the discovery of new therapeutics that more effectively target the unique aspects of each individual patient's TME. STATEMENT OF SIGNIFICANCE: In this article, we have presented the case for the application of biomaterials in developing personalized models of complex diseases such as cancers. TSM could bring about breakthroughs in the promise of precision medicine. The critical components of the diverse tumor microenvironments, that lead to treatment failures, include cellular- and extracellular matrix- heterogeneity, and biophysical signals to the cells. Therefore, we have described these dynamic components of the tumor microenvironments, and have highlighted how contemporary biomaterials can be utilized to create personalized in vitro models of cancers. We have also described the application of the TSM to predict the dynamic patterns of disease progression, and predict effective therapies that can produce durable responses, limit relapses, and treat any minimal residual disease.

The MEK1/2 Pathway as a Therapeutic Target in High-Grade Serous Ovarian Carcinoma.

High-grade serous ovarian carcinoma (HGSOC) is the deadliest of gynecological cancers due to its high recurrence rate and acquired chemoresistance. RAS/MEK/ERK pathway activation is linked to cell proliferation and therapeutic resistance, but the role of MEK1/2-ERK1/2 pathway in HGSOC is poorly investigated. We evaluated MEK1/2 pathway activity in clinical HGSOC samples and ovarian cancer cell lines using immunohistochemistry, immunoblotting, and RT-qPCR. HGSOC cell lines were used to assess immediate and lasting effects of MEK1/2 inhibition with trametinib in vitro. Trametinib effect on tumor growth in vivo was investigated using mouse xenografts. MEK1/2 pathway is hyperactivated in HGSOC and is further stimulated by cisplatin treatment. Trametinib treatment causes cell cycle arrest in G1/0-phase and reduces tumor growth rate in vivo but does not induce cell death or reduce fraction of CD133+ stem-like cells, while increasing expression of stemness-associated genes instead. Transient trametinib treatment causes long-term increase in a subpopulation of cells with high aldehyde dehydrogenase (ALDH)1 activity that can survive and grow in non-adherent conditions. We conclude that MEK1/2 inhibition may be a promising approach to suppress ovarian cancer growth as a maintenance therapy. Promotion of stem-like properties upon MEK1/2 inhibition suggests a possible mechanism of resistance, so a combination with CSC-targeting drugs should be considered.

Lysis and direct detection of coliforms on printed paper-based microfluidic devices.

Coliforms are one of the most common families of bacteria responsible for water contamination. Certain coliform strains can be extremely toxic, and even fatal if consumed. Current technologies for coliform detection are expensive, require multiple complicated steps, and can take up to 24 hours to produce accurate results. Recently, open-channel, paper-based microfluidic devices have become popular for rapid, inexpensive, and accurate bioassays. In this work, we have created an integrated microfluidic coliform lysis and detection device by fabricating customizable omniphilic regions via direct printing of omniphilic channels on an omniphobic, fluorinated paper. This paper-based device is the first of its kind to demonstrate successful cell lysing on-chip, as it can allow for the flow and control of both high and low surface tension liquids, including different cell lysing agents. The fabricated microfluidic device was able to successfully detect E. coli, via the presence of the coliform-specific enzyme, ß-galactosidase, at a concentration as low as ∼104 CFU mL-1. Further, E. coli at an initial concentration of 1 CFU mL-1 could be detected after only 6 hours of incubation. We believe that these devices can be readily utilized for real world E. coli contamination detection in multiple applications, including food and water safety.

Carcinoma-Associated Mesenchymal Stem Cells Promote Chemoresistance in Ovarian Cancer Stem Cells via PDGF Signaling.

Within the ovarian cancer tumor microenvironment, cancer stem-like cells (CSC) interact with carcinoma associated mesenchymal stem/stromal cells (CA-MSC) through multiple secreted cytokines and growth factors. These paracrine interactions have been revealed to cause enrichment of CSC and their chemoprotection; however, it is still not known if platelet-derived growth factor (PDGF) signaling is involved in facilitating these responses. In order to probe this undiscovered bidirectional communication, we created a model of ovarian malignant ascites in the three-dimensional (3D) hanging drop heterospheroid array, with CSC and CA-MSC. We hypothesized that PDGF secretion by CA-MSC increases self-renewal, migration, epithelial to mesenchymal transition (EMT) and chemoresistance in ovarian CSC. Our results indicate that PDGF signaling in the CSC-MSC heterospheroids significantly increased stemness, metastatic potential and chemoresistance of CSC. Knockdown of PDGFB in MSC resulted in abrogation of these phenotypes in the heterospheroids. Our studies also reveal a cross-talk between PDGF and Hedgehog signaling in ovarian cancer. Overall, our data suggest that when the stromal signaling via PDGF to ovarian CSC is blocked in addition to chemotherapy pressure, the tumor cells are significantly more sensitive to chemotherapy. Our results emphasize the importance of disrupting the signals from the microenvironment to the tumor cells, in order to improve response rates. These findings may lead to the development of combination therapies targeting stromal signaling (such as PDGF and Hedgehog) that can abrogate the tumorigenic, metastatic and platinum resistant phenotypes of ovarian CSC through additional investigations.

Compressive Stimulation Enhances Ovarian Cancer Proliferation, Invasion, Chemoresistance, and Mechanotransduction via CDC42 in a 3D Bioreactor.

This report investigates the role of compressive stress on ovarian cancer in a 3D custom built bioreactor. Cells within the ovarian tumor microenvironment experience a range of compressive stimuli that contribute to mechanotransduction. As the ovarian tumor expands, cells are exposed to chronic load from hydrostatic pressure, displacement of surrounding cells, and growth induced stress. External dynamic stimuli have been correlated with an increase in metastasis, cancer stem cell marker expression, chemoresistance, and proliferation in a variety of cancers. However, how these compressive stimuli contribute to ovarian cancer progression is not fully understood. In this report, high grade serous ovarian cancer cell lines were encapsulated within an ECM mimicking hydrogel comprising of agarose and collagen type I, and stimulated with confined cyclic or static compressive stresses for 24 and 72 h. Compression stimulation resulted in a significant increase in proliferation, invasive morphology, and chemoresistance. Additionally, CDC42 was upregulated in compression stimulated conditions, and was necessary to drive increased proliferation and chemoresistance. Inhibition of CDC42 lead to significant decrease in proliferation, survival, and increased chemosensitivity. In summary, the dynamic in vitro 3D platform developed in this report, is ideal for understanding the influence of compressive stimuli, and can be widely applicable to any epithelial cancers. This work reinforces the critical need to consider compressive stimulation in basic cancer biology and therapeutic developments.

Phase II clinical trial of metformin as a cancer stem cell-targeting agent in ovarian cancer.

BACKGROUNDEpidemiologic studies suggest that metformin has antitumor effects. Laboratory studies indicate metformin impacts cancer stem-like cells (CSCs). As part of a phase II trial, we evaluated the impact of metformin on CSC number and on carcinoma-associated mesenchymal stem cells (CA-MSCs) and clinical outcomes in nondiabetic patients with advanced-stage epithelial ovarian cancer (EOC).METHODSThirty-eight patients with stage IIC (n = 1)/III (n = 25)/IV (n = 12) EOC were treated with either (a) neoadjuvant metformin, debulking surgery, and adjuvant chemotherapy plus metformin or (b) neoadjuvant chemotherapy and metformin, interval debulking surgery, and adjuvant chemotherapy plus metformin. Metformin-treated tumors, compared with historical controls, were evaluated for CSC number and chemotherapy response. Primary endpoints were (a) a 2-fold or greater reduction in aldehyde dehydrogenase-positive (ALDH+) CD133+ CSCs and (b) a relapse-free survival at 18 months of more than 50%.RESULTSMetformin was well tolerated. Median progression-free survival was 18.0 months (95% CI 14.0-21.6) with relapse-free survival at 18 months of 59.3% (95% CI 38.6-70.5). Median overall survival was 57.9 months (95% CI 28.0-not estimable). Tumors treated with metformin had a 2.4-fold decrease in ALDH+CD133+ CSCs and increased sensitivity to cisplatin ex vivo. Furthermore, metformin altered the methylation signature in CA-MSCs, which prevented CA-MSC-driven chemoresistance in vitro.CONCLUSIONTranslational studies confirm an impact of metformin on EOC CSCs and suggest epigenetic change in the tumor stroma may drive the platinum sensitivity ex vivo. Consistent with this, metformin therapy was associated with better-than-expected overall survival, supporting the use of metformin in phase III studies.TRIAL REGISTRATIONClinicalTrials.gov NCT01579812.

Expansion of Circulating Tumor Cells from Patients with Locally Advanced Pancreatic Cancer Enable Patient Derived Xenografts and Functional Studies for Personalized Medicine.

Improvement in pancreatic cancer treatment represents an urgent medical goal that has been hampered by the lack of predictive biomarkers. Circulating Tumor Cells (CTCs) may be able to overcome this issue by allowing the monitoring of therapeutic response and tumor aggressiveness through ex vivo expansion. The successful expansion of CTCs is challenging, due to their low numbers in blood and the high abundance of blood cells. Here, we explored the utility of pancreatic CTC cultures as a preclinical model for treatment response. CTCs were isolated from ten patients with locally advanced pancreatic cancer using the Labyrinth, a biomarker independent, size based, inertial microfluidic separation device. Three patient-derived CTC samples were successfully expanded in adherent and spheroid cultures. Molecular and functional characterization was performed on the expanded CTC lines. CTC lines exhibited KRAS mutations, consistent with pancreatic cancers. Additionally, we evaluated take rate and metastatic potential in vivo and examined the utility of CTC lines for cytotoxicity assays. Patient derived expanded CTCs successfully generated patient derived xenograft (PDX) models with a 100% take rate. Our results demonstrate that CTC cultures are possible and provide a valuable resource for translational pancreatic cancer research, while also providing meaningful insight into the development of distant metastasis, as well as treatment resistance.

Physiologic Patient Derived 3D Spheroids for Anti-neoplastic Drug Screening to Target Cancer Stem Cells.

In this protocol, we outline the procedure for generation of tumor spheroids within 384-well hanging droplets to allow for high-throughput screening of anti-cancer therapeutics in a physiologically representative microenvironment. We outline the formation of patient derived cancer stem cell spheroids, as well as, the manipulation of these spheroids for thorough analysis following drug treatment. Specifically, we describe collection of spheroid morphology, proliferation, viability, drug toxicity, cell phenotype and cell localization data. This protocol focuses heavily on analysis techniques that are easily implemented using the 384-well hanging drop platform, making it ideal for high throughput drug screening. While we emphasize the importance of this model in ovarian cancer studies and cancer stem cell research, the 384-well platform is amenable to research of other cancer types and disease models, extending the utility of the platform to many fields. By improving the speed of personalized drug screening and the quality of screening results through easily implemented physiologically representative 3D cultures, this platform is predicted to aid in the development of new therapeutics and patient-specific treatment strategies, and thus have wide-reaching clinical impact.

The Role of Cancer Stem Cells and Mechanical Forces in Ovarian Cancer Metastasis.

Ovarian cancer is an extremely lethal gynecologic disease; with the high-grade serous subtype predominantly associated with poor survival rates. Lack of early diagnostic biomarkers and prevalence of post-treatment recurrence, present substantial challenges in treating ovarian cancers. These cancers are also characterized by a high degree of heterogeneity and protracted metastasis, further complicating treatment. Within the ovarian tumor microenvironment, cancer stem-like cells and mechanical stimuli are two underappreciated key elements that play a crucial role in facilitating these outcomes. In this review article, we highlight their roles in modulating ovarian cancer metastasis. Specifically, we outline the clinical relevance of cancer stem-like cells, and challenges associated with their identification and characterization and summarize the ways in which they modulate ovarian cancer metastasis. Further, we review the mechanical cues in the ovarian tumor microenvironment, including, tension, shear, compression and matrix stiffness, that influence (cancer stem-like cells and) metastasis in ovarian cancers. Lastly, we outline the challenges associated with probing these important modulators of ovarian cancer metastasis and provide suggestions for incorporating these cues in basic biology and translational research focused on metastasis. We conclude that future studies on ovarian cancer metastasis will benefit from the careful consideration of mechanical stimuli and cancer stem cells, ultimately allowing for the development of more effective therapies.