Macrophage Checkpoint Nanoimmunotherapy Has the Potential to Reduce Malignant Progression in Bioengineered In Vitro Models of Ovarian Cancer.

Most ovarian carcinoma (OvCa) patients present with advanced disease at the time of diagnosis. Malignant, metastatic OvCa is invasive and has poor prognosis, exposing the need for improved therapeutic targeting. High CD47 (OvCa) and SIRPα (macrophage) expression has been linked to decreased survival, making this interaction a significant target for therapeutic discovery. Even so, previous attempts have fallen short, limited by CD47 antibody specificity and efficacy. Macrophages are an important component of the OvCa tumor microenvironment and are manipulated to aid in cancer progression via CD47-SIRPα signaling. Thus, we have leveraged lipid-based nanoparticles (LNPs) to design a therapy uniquely situated to home to phagocytic macrophages expressing the SIRPα protein in metastatic OvCa. CD47-SIRPα presence was evaluated in patient histological sections using immunohistochemistry. 3D tumor spheroids generated on a hanging drop array with OVCAR3 high-grade serous OvCa and THP-1-derived macrophages created a representative model of cellular interactions involved in metastatic OvCa. Microfluidic techniques were employed to generate LNPs encapsulating SIRPα siRNA (siSIRPα) to affect the CD47-SIRPα signaling between the OvCa and macrophages. siSIRPα LNPs were characterized for optimal size, charge, and encapsulation efficiency. Uptake of the siSIRPα LNPs by macrophages was assessed by Incucyte. Following 48 h of 25 nM siSIRPα treatment, OvCa/macrophage heterospheroids were evaluated for SIRPα knockdown, platinum chemoresistance, and invasiveness. OvCa patient tumors and in vitro heterospheroids expressed CD47 and SIRPα. Macrophages in OvCa spheroids increased carboplatin resistance and invasion, indicating a more malignant phenotype. We observed successful LNP uptake by macrophages causing significant reduction in SIRPα gene and protein expressions and subsequent reversal of pro-tumoral alternative activation. Disrupting CD47-SIRPα interactions resulted in sensitizing OvCa/macrophage heterospheroids to platinum chemotherapy and reversal of cellular invasion outside of heterospheroids. Ultimately, our results strongly indicate the potential of using LNP-based nanoimmunotherapy to reduce malignant progression of ovarian cancer.

Advances in cancer mechanobiology: Metastasis, mechanics, and materials.

Within the tumor microenvironment (TME), tumor cells are exposed to numerous mechanical forces, both internally and externally, which contribute to the metastatic cascade. From the initial growth of the tumor to traveling through the vasculature and to the eventual colonization of distant organs, tumor cells are continuously interacting with their surroundings through physical contact and mechanical force application. The mechanical forces found in the TME can be simplified into three main categories: (i) shear stress, (ii) tension and strain, and (iii) solid stress and compression. Each force type can independently impact tumor growth and progression. Here, we review recent bioengineering strategies, which have been employed to establish the connection between mechanical forces and tumor progression. While many cancers are explored in this review, we place great emphasis on cancers that are understudied in their response to mechanical forces, such as ovarian and colorectal cancers. We discuss the major steps of metastatic transformation and present novel, recent advances in model systems used to study how mechanical forces impact the study of the metastatic cascade. We end by summarizing systems that incorporate multiple forces to expand the complexity of our understanding of how tumor cells sense and respond to mechanical forces in their environment. Future studies would also benefit from the inclusion of time or the aspect of mechanical memory to further enhance this field. While the knowledge of mechanical forces and tumor metastasis grows, developing novel materials and in vitro systems are essential to providing new insight into predicting, treating, and preventing cancer progression and metastasis.

Degrees of macrophage-facilitated healing in aneurysm occlusion devices.

Insufficient healing of aneurysms following treatment with vascular occlusion devices put patients at severe risk of fatal rupture. Therefore, promoting healing and not just occlusion is vital to enhance aneurysm healing. Following occlusion device implantation, healing is primarily orchestrated by macrophage immune cells, ending with fibroblasts depositing collagen to stabilize the aneurysm neck and dome, preventing rupture. Several modified occlusion devices are available currently on-market. Previous in vivo work demonstrated that modifications of occlusion devices with a shape memory polymer foam had enhanced aneurysm healing outcomes. To better understand cellular response to occlusion devices and improve aneurysm occlusion device design variables, we developed an in vitro assay to isolate prominent interactions between devices and key healing players: macrophages and fibroblasts. We used THP-1 monocyte derived macrophages and human dermal fibroblasts in our cell culture models. Macrophages were allowed device contact with on-market competitor aneurysm occlusion devices for up to 96 h, to allow for any spontaneous device-driven macrophage activation. Macrophage secreted factors were captured in the culture media, in response to device-specific activation. Fibroblasts were then exposed to device-conditioned macrophage media (with secreted factors alone), to determine if there were any device-induced changes in collagen secretion. Our in vitro studies were designed to test the direct effect of devices on macrophage activation, and the indirect effect of devices on collagen secretion by fibroblasts to promote aneurysm healing and stabilization. Over 96 h, macrophages displayed significant migration toward and interaction with all tested devices. As compared to other devices, shape memory polymer foams (SMM, Shape Memory Medical) induced significant changes in gene expression indicating a shift toward an anti-inflammatory pro-healing M2-like phenotype. Similarly, macrophages in contact with SMM devices secreted more vascular endothelial growth factor (VEGF) compared with other devices. Macrophage conditioned media from SMM-contacted macrophages actively promoted fibroblast secretion of collagen, comparable to amounts observed with exogenous stimulation via VEGF supplementation. Our data indicate that SMM devices may promote good aneurysm healing outcomes, because collagen production is an essential step to ultimately stabilize an aneurysm.

Peristalsis-Associated Mechanotransduction Drives Malignant Progression of Colorectal Cancer.

Introduction: In the colorectal cancer (CRC) tumor microenvironment, cancerous and precancerous cells continuously experience mechanical forces associated with peristalsis. Given that mechanical forces like shear stress and strain can positively impact cancer progression, we explored the hypothesis that peristalsis may also contribute to malignant progression in CRC. We defined malignant progression as enrichment of cancer stem cells and the acquisition of invasive behaviors, both vital to CRC progression. Methods: We leveraged our peristalsis bioreactor to expose CRC cell lines (HCT116), patient-derived xenograft (PDX1,2) lines, or non-cancerous intestinal cells (HIEC-6) to forces associated with peristalsis in vitro. Cells were maintained in static control conditions or exposed to peristalsis for 24 h prior to assessment of cancer stem cell (CSC) emergence or the acquisition of invasive phenotypes. Results: Exposure of HCT116 cells to peristalsis significantly increased the emergence of LGR5+ CSCs by 1.8-fold compared to static controls. Peristalsis enriched LGR5 positivity in several CRC cell lines, notably significant in KRAS mutant lines. In contrast, peristalsis failed to increase LGR5+ in non-cancerous intestinal cells, HIEC-6. LGR5+ emergence downstream of peristalsis was dependent on ROCK and Wnt activity, and not YAP1 activation. Additionally, HCT116 cells adopted invasive morphologies when exposed to peristalsis, with increased filopodia density and epithelial to mesenchymal gene expression, in a Wnt dependent manner. Conclusions: Peristalsis associated forces drive malignant progression of CRC via ROCK, YAP1, and Wnt-related mechanotransduction. Supplementary Information: The online version contains supplementary material available at 10.1007/s12195-023-00776-w.

Molecular portraits of cell cycle checkpoint kinases in cancer evolution, progression, and treatment responsiveness.

Cell cycle dysregulation is prerequisite for cancer formation. However, it is unknown whether the mode of dysregulation affects disease characteristics. Here, we conduct comprehensive analyses of cell cycle checkpoint dysregulation using patient data and experimental investigations. We find that ATM mutation predisposes the diagnosis of primary estrogen receptor (ER)+/human epidermal growth factor (HER)2- cancer in older women. Conversely, CHK2 dysregulation induces formation of metastatic, premenopausal ER+/HER2- breast cancer (P = 0.001) that is treatment-resistant (HR = 6.15, P = 0.01). Lastly, while mutations in ATR alone are rare, ATR/TP53 co-mutation is 12-fold enriched over expected in ER+/HER2- disease (P = 0.002) and associates with metastatic progression (HR = 2.01, P = 0.006). Concordantly, ATR dysregulation induces metastatic phenotypes in TP53 mutant, not wild-type, cells. Overall, we identify mode of cell cycle dysregulation as a distinct event that determines subtype, metastatic potential, and treatment responsiveness, providing rationale for reconsidering diagnostic classification through the lens of the mode of cell cycle dysregulation..

The Proteolytic Landscape of Ovarian Cancer: Applications in Nanomedicine.

Ovarian cancer (OvCa) is one of the leading causes of mortality globally with an overall 5-year survival of 47%. The predominant subtype of OvCa is epithelial carcinoma, which can be highly aggressive. This review launches with a summary of the clinical features of OvCa, including staging and current techniques for diagnosis and therapy. Further, the important role of proteases in OvCa progression and dissemination is described. Proteases contribute to tumor angiogenesis, remodeling of extracellular matrix, migration and invasion, major processes in OvCa pathology. Multiple proteases, such as metalloproteinases, trypsin, cathepsin and others, are overexpressed in the tumor tissue. Presence of these catabolic enzymes in OvCa tissue can be exploited for improving early diagnosis and therapeutic options in advanced cases. Nanomedicine, being on the interface of molecular and cellular scales, can be designed to be activated by proteases in the OvCa microenvironment. Various types of protease-enabled nanomedicines are described and the studies that focus on their diagnostic, therapeutic and theranostic potential are reviewed.

Decellularized organ biomatrices facilitate quantifiable in vitro 3D cancer metastasis models.

Metastatic cancers are chemoresistant, involving complex interplay between disseminated cancer cell aggregates and the distant organ microenvironment (extracellular matrix and stromal cells). Conventional metastasis surrogates (scratch/wound healing, Transwell migration assays) lack 3D architecture and ECM presence. Metastasis studies can therefore significantly benefit from biomimetic 3D in vitro models recapitulating the complex cascade of distant organ invasion and colonization by collective clusters of cells. We aimed to engineer reproducible and quantifiable 3D models of highly therapy-resistant cancer processes: (i) colorectal cancer liver metastasis; and (ii) breast cancer lung metastasis. Metastatic seeds are engineered using 3D tumor spheroids to recapitulate the 3D aggregation of cancer cells both in the tumor and in circulation throughout the metastatic cascade of many cancers. Metastatic soil was engineered by decellularizing porcine livers and lungs to generate biomatrix scaffolds, followed by extensive materials characterization. HCT116 colorectal and MDA-MB-231 breast cancer spheroids were generated on hanging drop arrays to initiate clustered metastatic seeding into liver and lung biomatrix scaffolds, respectively. Between days 3-7, biomatrix cellular colonization was apparent with increased metabolic activity and the presence of cellular nests evaluated via multiphoton microscopy. HCT116 and MDA-MB-231 cells colonized liver and lung biomatrices, and at least 15% of the cells invaded more than 20 µm from the surface. Engineered metastases also expressed increased signatures of genes associated with the metastatic epithelial to mesenchymal transition (EMT). Importantly, inhibition of matrix metalloproteinase-9 inhibited metastatic invasion into the biomatrix. Furthermore, metastatic nests were significantly more chemoresistant (>3 times) to the anti-cancer drug oxaliplatin, compared to 3D spheroids. Together, our data indicated that HCT116 and MDA-MB-231 spheroids invade, colonize, and proliferate in livers and lungs establishing metastatic nests in 3D settings in vitro. The metastatic nature of these cells was confirmed with functional readouts regarding EMT and chemoresistance. Modeling the dynamic metastatic cascade in vitro has potential to identify therapeutic targets to treat or prevent metastatic progression in chemoresistant metastatic cancers.

How inclusive are cell lines in preclinical engineered cancer models?

Diverse factors contribute to significant and dire disparities in cancer risk and treatment outcomes. To address this, there was a call for inclusion of sex as a biological variable, which resulted in more instances of careful inclusion of sex in preclinical studies of cancer. Another variable in cancer treatment is genetic ancestry. Although this is considered explicitly in clinical research, it is considerably neglected in preclinical studies. Preclinical research can use several 3D in vitro model systems, such as spheroids/organoids, xenografts, or other bioengineered systems that combine biomaterials and cellular material. Ultimately, the cellular base for all of these in vitro model systems is derived from human cell lines or patient samples, to investigate mechanisms of cancer and screen novel therapeutics, all of which aim to maximize successful outcomes in clinical trials. This in itself offers an opportunity to potentiate effective treatments for many groups of people, when diverse variables like genetic ancestry are consciously included into study design. This Perspective highlights the need for conscious inclusion of genetic ancestry in preclinical cancer tissue engineering, especially when it pertains to determining therapeutic outcomes.

The Future of ctDNA-Defined Minimal Residual Disease: Personalizing Adjuvant Therapy in Colorectal Cancer.

Our understanding of the diagnostic and prognostic use of circulating tumor DNA (ctDNA) in colorectal cancer (CRC) has broadly expanded over the past few years. The utilization of ctDNA to detect minimal residual disease is currently being employed across the continuum of cancer care. The lead-time of ctDNA positivity to radiographic recurrence in stage I to III CRC is up to 9 months on average, which provides a therapeutic window for a group of high-risk patients who will ultimately recur. There are several ongoing prospective clinical trials that investigate whether ctDNA can be used as an integral biomarker to risk stratify CRC patients and guide adjuvant treatment decisions. In this review, we summarize the evidence supporting the promise of ctDNA-defined MRD in CRC and highlight the current ctDNA guided adjuvant prospective clinical trials.

Modeling of Nanotherapy Response as a Function of the Tumor Microenvironment: Focus on Liver Metastasis.

The tumor microenvironment (TME) presents a challenging barrier for effective nanotherapy-mediated drug delivery to solid tumors. In particular for tumors less vascularized than the surrounding normal tissue, as in liver metastases, the structure of the organ itself conjures with cancer-specific behavior to impair drug transport and uptake by cancer cells. Cells and elements in the TME of hypovascularized tumors play a key role in the process of delivery and retention of anti-cancer therapeutics by nanocarriers. This brief review describes the drug transport challenges and how they are being addressed with advanced in vitro 3D tissue models as well as with in silico mathematical modeling. This modeling complements network-oriented techniques, which seek to interpret intra-cellular relevant pathways and signal transduction within cells and with their surrounding microenvironment. With a concerted effort integrating experimental observations with computational analyses spanning from the molecular- to the tissue-scale, the goal of effective nanotherapy customized to patient tumor-specific conditions may be finally realized.

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.

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.

Engineered 3D Model of Cancer Stem Cell Enrichment and Chemoresistance.

Intraperitoneal dissemination of ovarian cancers is preceded by the development of chemoresistant tumors with malignant ascites. Despite the high levels of chemoresistance and relapse observed in ovarian cancers, there are no in vitro models to understand the development of chemoresistance in situ. METHOD: We describe a highly integrated approach to establish an in vitro model of chemoresistance and stemness in ovarian cancer, using the 3D hanging drop spheroid platform. The model was established by serially passaging non-adherent spheroids. At each passage, the effectiveness of the model was evaluated via measures of proliferation, response to treatment with cisplatin and a novel ALDH1A inhibitor. Concomitantly, the expression and tumor initiating capacity of cancer stem-like cells (CSCs) was analyzed. RNA-seq was used to establish gene signatures associated with the evolution of tumorigenicity, and chemoresistance. Lastly, a mathematical model was developed to predict the emergence of CSCs during serial passaging of ovarian cancer spheroids. RESULTS: Our serial passage model demonstrated increased cellular proliferation, enriched CSCs, and emergence of a platinum resistant phenotype. In vivo tumor xenograft assays indicated that later passage spheroids were significantly more tumorigenic with higher CSCs, compared to early passage spheroids. RNA-seq revealed several gene signatures supporting the emergence of CSCs, chemoresistance, and malignant phenotypes, with links to poor clinical prognosis. Our mathematical model predicted the emergence of CSC populations within serially passaged spheroids, concurring with experimentally observed data. CONCLUSION: Our integrated approach illustrates the utility of the serial passage spheroid model for examining the emergence and development of chemoresistance in ovarian cancer in a controllable and reproducible format.

Ovarian cancer stem cells and macrophages reciprocally interact through the WNT pathway to promote pro-tumoral and malignant phenotypes in 3D engineered microenvironments.

BACKGROUND: Innate immune cells such as macrophages are abundantly present within malignant ascites, where they share the microenvironment with ovarian cancer stem cells (CSC). METHODS: To mimic this malignant ascites microenvironment, we created a hanging-drop hetero-spheroid model to bring CSCs and macrophages in close association. Within these hetero-spheroids, CD68+ macrophages (derived from U937 or peripheral blood monocytes) make up ~ 20% of the population, while the rest are ovarian cancer cells and ovarian cancer stem cells (derived from the high grade serous ovarian cancer cell line, OVCAR3). RESULTS: Our results indicate that CSCs drive the upregulation of M2 macrophage marker CD206 within hetero-spheroids, compared to bulk ovarian cancer cells, implying an inherently more immuno-suppressive program. Moreover, an increased maintenance of elevated aldehyde dehydrogenase (ALDH) activity is noted within hetero-spheroids that include pre-polarized CD206+ M2 macrophages, implying a reciprocal interaction that drives pro-tumoral activation as well as CSC self-renewal. Consistent with enriched CSCs, we also observe increased levels of pro-tumoral IL-10 and IL-6 cytokines in the CSC/M2-macrophage hetero-spheroids. CSC/M2-macrophage hetero-spheroids are also less sensitive to the chemotherapeutic agent carboplatin and are subsequently more invasive in transwell assays. Using inhibitors of WNT secretion in both CSCs and macrophages, we found that CSC-derived WNT ligands drove CD206+ M2 macrophage activation, and that, conversely, macrophage-derived WNT ligands enriched ALDH+ cells within the CSC compartment of hetero-spheroids. Upon examination of specific WNT ligand expression within the monocyte-derived macrophage system, we observed a significant elevation in gene expression for WNT5B. In CSCs co-cultured with macrophages within hetero-spheroids, increases in several WNT ligands were observed, and this increase was significantly inhibited when WNT5B was knocked down in macrophages. CONCLUSIONS: Our data implies that macrophage- initiated WNT signaling could play a significant role in the maintenance of stemness, and the resulting phenotypes of chemoresistance and invasiveness. Our results indicate paracrine WNT activation during CSC/M2 macrophages interaction constitutes a positive feedback loop that likely contributes to the more aggressive phenotype, which makes the WNT pathway a potential target to reduce the CSC and M2 macrophage compartments in the tumor microenvironment.

Integrated cancer tissue engineering models for precision medicine.

Tumors are not merely cancerous cells that undergo mindless proliferation. Rather, they are highly organized and interconnected organ systems. Tumor cells reside in complex microenvironments in which they are subjected to a variety of physical and chemical stimuli that influence cell behavior and ultimately the progression and maintenance of the tumor. As cancer bioengineers, it is our responsibility to create physiologic models that enable accurate understanding of the multi-dimensional structure, organization, and complex relationships in diverse tumor microenvironments. Such models can greatly expedite clinical discovery and translation by closely replicating the physiological conditions while maintaining high tunability and control of extrinsic factors. In this review, we discuss the current models that target key aspects of the tumor microenvironment and their role in cancer progression. In order to address sources of experimental variation and model limitations, we also make recommendations for methods to improve overall physiologic reproducibility, experimental repeatability, and rigor within the field. Improvements can be made through an enhanced emphasis on mathematical modeling, standardized in vitro model characterization, transparent reporting of methodologies, and designing experiments with physiological metrics. Taken together these considerations will enhance the relevance of in vitro tumor models, biological understanding, and accelerate treatment exploration ultimately leading to improved clinical outcomes. Moreover, the development of robust, user-friendly models that integrate important stimuli will allow for the in-depth study of tumors as they undergo progression from non-transformed primary cells to metastatic disease and facilitate translation to a wide variety of biological and clinical studies.

Bioengineering and regeneration of gastrointestinal tissue: where are we now and what comes next?

INTRODUCTION: The field of tissue engineering and regenerative medicine has been applied to the gastrointestinal (GI) tract for a couple decades. Several achievements have been accomplished that provide promising tools for treating diseases of the GI tract. AREAS COVERED: The work described in this review covers the traditional aspect of using cells and scaffolds to replace parts of the tract. Several studies investigated different types of biomaterials and different types of cells. A more recent approach involved the use of gut-derived organoid units that can differentiate into all gut cell layers. The most recent approach introduced the use of organ-on-a-chip concept to understand the physiology and pathophysiology of the GI system. EXPERT OPINION: The different approaches tackle the diseases of the GI tract from different perspectives. While all these different approaches provide a promising and encouraging future for this field, the translational aspect is yet to be studied.

Self-Renewal and CSCs In Vitro Enrichment: Growth as Floating Spheres.

Cancer stem cells (CSC) are a vital component to the progression and reoccurrence of cancers, making them a primary target of study for both fundamental understanding of cancer biology and the development of effective and targeted treatments. CSCs reside in a complex 3D microenvironment, and the 3D spheroids are an indispensable tool in tumor biology due to their 3D structure and replication of the tumor microenvironment. Within this chapter the methodology for CSC isolation, suspension culture in hanging drop model, and characterization assays for CSC are described. First, the methodology for identifying and isolating CSCs from patient tumors, ascites, or cancer cell lines is described through the use of FACS analysis. Next, a detailed description of 3D hanging drop model for generating CSC spheroids is provided, followed by maintenance and monitoring techniques for extended 3D culture. Analysis methods are described for the quantification of CSC spheroid proliferation and viability tracking, throughout culture by on-plate alamarBlue fluorescence. Additional viability assays are described utilizing confocal microscopy with Live/Dead Viability/Cytotoxicity Kit. The characterization of CSCs populations within spheroids is described through FACS analysis. Further, an immunohistochemistry procedure is described for cell-cell and cell-matrix interaction assessment. Finally, several notes and tips for successful experiments with 3D CSC spheroids on the hanging drop model are provided. These methods are not only applicable to CSCs within a variety of tumor cell types, for not only understanding the fundamental tumor biology, but also for drug screening and development of preclinical chemotherapeutic strategies.

Printing of small molecular medicines from the vapor phase.

There is growing need to develop efficient methods for early-stage drug discovery, continuous manufacturing of drug delivery vehicles, and ultra-precise dosing of high potency drugs. Here we demonstrate the use of solvent-free organic vapor jet printing to deposit nanostructured films of small molecular pharmaceutical ingredients, including caffeine, paracetamol, ibuprofen, tamoxifen, BAY 11-7082 and fluorescein, with accuracy on the scale of micrograms per square centimeter, onto glass, Tegaderm, Listerine tabs, and stainless steel microneedles. The printed films exhibit similar crystallographic order and chemistry as the original powders; controlled, order-of-magnitude enhancements of dissolution rate are observed relative to powder-form particles. In vitro treatment of breast and ovarian cancer cell cultures in aqueous media by tamoxifen and BAY 11-7082 films shows similar behavior to drugs pre-dissolved in dimethyl sulfoxide. The demonstrated precise printing of medicines as films, without the use of solvents, can accelerate drug screening and enable continuous manufacturing, while enhancing dosage accuracy.Traditional approaches used in the pharmaceutical industry are not precise or versatile enough for customized medicine formulation and manufacture. Here the authors produce a method to form coatings, with accurate dosages, as well as a means of closely controlling dissolution kinetics.

Personalized Medicine-Based Approach to Model Patterns of Chemoresistance and Tumor Recurrence Using Ovarian Cancer Stem Cell Spheroids.

Purpose: Chemoresistant ovarian cancers grow in suspension within the ascites fluid. To screen the effect of chemotherapeutics and biologics on resistant ovarian cancers with a personalized basis, we developed a 3D hanging drop spheroid platform.Experimental Design: We initiated spheroids with primary aldehyde dehydrogenase-positive (ALDH+) CD133+ ovarian cancer stem cells (OvCSC) from different patient samples and demonstrated that stem cell progeny from harvested spheroids was similar to the primary tumor. OvCSC spheroids were utilized to initiate tumors in immunodeficient mice. Drug responses to cisplatin and ALDH-targeting compound or JAK2 inhibitor determined whether the OvCSC population within the spheroids could be targeted. Cells that escaped therapy were isolated and used to initiate new spheroids and model tumor reemergence in a personalized manner.Results: OvCSC spheroids from different patients exhibited varying and personalized responses to chemotherapeutics. Xenografts were established from OvCSC spheroids, even with a single spheroid. Distinct responses to therapy were observed in distinct primary tumor xenografts similar to those observed in spheroids. Spheroids resistant to cisplatin/ALDH inhibitor therapy had persistent, albeit lower ALDH expression and complete loss of CD133 expression, whereas those resistant to cisplatin/JAK2 inhibitor therapy were enriched for ALDH+ cells.Conclusions: Our 3D hanging drop suspension platform can be used to propagate primary OvCSCs that represent individual patient tumors effectively by differentiating in vitro and initiating tumors in mice. Therefore, our platform can be used to study cancer stem cell biology and model tumor reemergence to identify new targeted therapeutics from an effective personalized medicine standpoint. Clin Cancer Res; 23(22); 6934-45. ©2017 AACR.