Identification and characterization of bladder cancer by low‐resolution fiber‐optic Raman spectroscopy

Raman spectroscopy has been proved to be a promising diagnostic technique for various cancers detection. A major drawback for its clinical translation is the intrinsic weakness of Raman effects. Highly sensitive equipment and optimal measurement conditions are generally applied to overcome this drawback. However, these equipment are usually bulky, expensive and may also be easily influenced by surrounding environment. In this preliminary work, a low‐resolution fiber‐optic Raman sensing system is applied to evaluate the diagnostic potential of Raman spectroscopy to identify different bladder pathologies ex vivo. A total number of 262 spectra taken from 32 bladder specimens are included in this study. These spectra are categorized into 3 groups by histopathological analysis, namely normal bladder tissues, low‐grade bladder tumors and high‐grade bladder tumors. Principal component analysis fed artificial neural network are used to train a classification model for the spectral data with 10‐fold cross‐validation and an overall prediction accuracy of 93.1% is obtained. The sensitivities and specificities for normal bladder tissues, low‐grade bladder tumors and high‐grade bladder tumors are 88.5% and 95.1%, 90.3% and 98%, and 97.5% and 96.4%, respectively. These results demonstrate the potential of using a low‐resolution fiber‐optic Raman system for in vivo bladder cancer diagnosis.      

The use of 3-D cultures for high-throughput screening: the multicellular spheroid model

Over the past few years, establishment and adaptation of cell-based assays for drug development and testing has become an important topic in high-throughput screening (HTS). Most new assays are designed to rapidly detect specific cellular effects reflecting action at various targets. However, although more complex than cell-free biochemical test systems, HTS assays using monolayer or suspension cultures still reflect a highly artificial cellular environment and may thus have limited predictive value for the clinical efficacy of a compound. Today's strategies for drug discovery and development, be they hypothesis free or mechanism based, require facile, HTS-amenable test systems that mimic the human tissue environment with increasing accuracy in order to optimize preclinical and preanimal selection of the most active molecules from a large pool of potential effectors, for example, against solid tumors. Indeed, it is recognized that 3-dimensional cell culture systems better reflect the in vivo behavior of most cell types. However, these 3-D test systems have not yet been incorporated into mainstream drug development operations. This article addresses the relevance and potential of 3-D in vitro systems for drug development, with a focus on screening for novel antitumor drugs. Examples of 3-D cell models used in cancer research are given, and the advantages and limitations of these systems of intermediate complexity are discussed in comparison with both 2-D culture and in vivo models. The most commonly used 3-D cell culture systems, multicellular spheroids, are emphasized due to their advantages and potential for rapid development as HTS systems. Thus, multicellular tumor spheroids are an ideal basis for the next step in creating HTS assays, which are predictive of in vivo antitumor efficacy.     

Doxorubicin kinetics and effects on lung cancer cell lines using in vitro Raman micro‐spectroscopy: binding signatures,drug resistance and DNA repair

Raman micro‐spectroscopy is a non‐invasive analytical tool, whose potential in cellular analysis and monitoring drug mechanisms of action has already been demonstrated, and which can potentially be used in pre‐clinical and clinical applications for the prediction of chemotherapeutic efficacy. To further investigate such potential clinical application, it is important to demonstrate its capability to differentiate drug mechanisms of action and cellular resistances. Using the example of Doxorubicin (DOX), in this study, it was used to probe the cellular uptake, signatures of chemical binding and subsequent cellular responses, of the chemotherapeutic drug in two lung cancer cell lines, A549 and Calu‐1. Multivariate statistical analysis was used to elucidate the spectroscopic signatures associated with DOX uptake and subcellular interaction. Biomarkers related to DNA damage and repair, and mechanisms leading to apoptosis were also measured and correlated to Raman spectral profiles. Results confirm the potential of Raman spectroscopic profiling to elucidate both drug kinetics and pharmacodynamics and differentiate cellular drug resistance associated with different subcellular accumulation rates and subsequent cellular response to DNA damage, pointing towards a better understanding of drug resistance for personalised targeted treatment.

     

Study of neuroprotective function of Ginkgo biloba extract (EGb761) derived‐flavonoid monomers using a three‐dimensional stem cell‐derived neural model

An in vitro three‐dimensional (3D) cell culture system that can mimic organ and tissue structure and function in vivo will be of great benefit for drug discovery and toxicity testing. In this study, the neuroprotective properties of the three most prevalent flavonoid monomers extracted from EGb 761 (isorharmnetin, kaempferol, and quercetin) were investigated using the developed 3D stem cell‐derived neural co‐culture model. Rat neural stem cells were differentiated into co‐culture of both neurons and astrocytes at an equal ratio in the developed 3D model and standard two‐dimensional (2D) model using a two‐step differentiation protocol for 14 days. The level of neuroprotective effect offered by each flavonoid was found to be aligned with its effect as an antioxidant and its ability to inhibit Caspase‐3 activity in a dose‐dependent manner. Cell exposure to quercetin (100 µM) following oxidative insult provided the highest levels of neuroprotection in both 2D and 3D models, comparable with exposure to 100 µM of Vitamin E, whilst exposure to isorhamnetin and kaempferol provided a reduced level of neuroprotection in both 2D and 3D models. At lower dosages (10 µM flavonoid concentration), the 3D model was more representative of results previously reported in vivo. The co‐cultures of stem cell derived neurons and astrocytes in 3D hydrogel scaffolds as an in vitro neural model closely replicates in vivo results for routine neural drug toxicity and efficacy testing. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:735–744, 2016     

Recapitulation of Tumor Heterogeneity and Molecular Signatures in a 3D Brain Cancer Model with Decreased Sensitivity to Histone Deacetylase Inhibition

Introduction

Physiologically relevant pre-clinical ex vivo models recapitulating CNS tumor micro-environmental complexity will aid development of biologically-targeted agents. We present comprehensive characterization of tumor aggregates generated using the 3D Rotary Cell Culture System (RCCS).

Methods

CNS cancer cell lines were grown in conventional 2D cultures and the RCCS and comparison with a cohort of 53 pediatric high grade gliomas conducted by genome wide gene expression and microRNA arrays, coupled with immunohistochemistry, ex vivo magnetic resonance spectroscopy and drug sensitivity evaluation using the histone deacetylase inhibitor, Vorinostat.

Results

Macroscopic RCCS aggregates recapitulated the heterogeneous morphology of brain tumors with a distinct proliferating rim, necrotic core and oxygen tension gradient. Gene expression and microRNA analyses revealed significant differences with 3D expression intermediate to 2D cultures and primary brain tumors. Metabolic profiling revealed differential profiles, with an increase in tumor specific metabolites in 3D. To evaluate the potential of the RCCS as a drug testing tool, we determined the efficacy of Vorinostat against aggregates of U87 and KNS42 glioblastoma cells. Both lines demonstrated markedly reduced sensitivity when assaying in 3D culture conditions compared to classical 2D drug screen approaches.

Conclusions

Our comprehensive characterization demonstrates that 3D RCCS culture of high grade brain tumor cells has profound effects on the genetic, epigenetic and metabolic profiles of cultured cells, with these cells residing as an intermediate phenotype between that of 2D cultures and primary tumors. There is a discrepancy between 2D culture and tumor molecular profiles, and RCCS partially re-capitulates tissue specific features, allowing drug testing in a more relevant ex vivo system.     

An in vitro study of the interaction of the chemotherapeutic drug Actinomycin D with lung cancer cell lines using Raman micro‐spectroscopy

The applications of Raman microspectroscopy have been extended in recent years into the field of clinical medicine, and specifically in cancer research, as a non‐invasive diagnostic method in vivo and ex vivo, and the field of pharmaceutical development as a label‐free predictive technique for new drug mechanisms of action in vitro. To further illustrate its potential for such applications, it is important to establish its capability to fingerprint drug mechanisms of action and different cellular reactions. In this study, cytotoxicity assays were employed to establish the toxicity profiles for 48 and 72 hours exposure of lung cancer cell lines, A549 and Calu‐1, after exposure to Actinomycin D (ACT) and Raman micro‐spectroscopy was used to track its mechanism of action at subcellular level and subsequent cellular responses. Multivariate data analysis was used to elucidate the spectroscopic signatures associated with ACT chemical binding and cellular resistances. Results show that the ACT uptake and mechanism of action are similar in the 2 cell lines, while A549 cells exhibits spectral signatures of resistance to apoptosis related to its higher chemoresistance to the anticancer drug ACT. The observations are discussed in comparison to previous studies of the similar anthracyclic chemotherapeutic agent Doxorubicin. A, Preprocessed Raman spectrum of ACT stock solution dissolved in sterile water and mean spectrum with SD of (B) nucleolus, (C) nucleus and (D) cytoplasm of A549 cell lines after 48 hours exposure to the corresponding IC₅₀.      

Differentiating responses of lung cancer cell lines to Doxorubicin exposure: in vitro Raman micro spectroscopy,oxidative stress and bcl‐2 protein expression

The potential of Raman micro spectroscopy as an in vitro, non‐invasive tool for clinical applications has been demonstrated in recent years, specifically for cancer research. To further illustrate its potential as a high content and label free technique, it is important to show its capability to elucidate drug mechanisms of action and cellular resistances. In this study, cytotoxicity assays were employed to establish the toxicity profiles for 24 hr exposure of lung cancer cell lines, A549 and Calu‐1, to the commercially available drug, doxorubicin (DOX). Raman spectroscopy, coupled with Confocal Laser Scanning Microscopy and Flow Cytometry, was used to track the DOX mechanism of action, at a subcellular level, and to study the mechanisms of cellular resistance to DOX. Biomarkers related to the drug mechanism of action and cellular resistance to apoptosis, namely reactive oxygen species (ROS) and bcl‐2 protein expression, respectively, were also measured and correlated to Raman spectral profiles. Calu‐1 cells are shown to exhibit spectroscopic signatures of both direct DNA damage due to intercalation in the nucleus and indirect damage due to oxidative stress in the cytoplasm, whereas the A549 cell line only exhibits signatures of the former mechanism of action.

PCA of nucleolar, nuclear and cytoplasmic regions of A549 and Calu‐1 with corresponding loadings of PC1 and PC2     

Cell classification with low‐resolution Raman spectroscopy (LRRS)

The identification of individual eukaryotic and prokaryotic cells is the backbone of clinical pathology and provides crucial information about the genesis and progression of a disease. While most commonly fluorescent‐label based methods are applied, label‐free methods, such as Raman spectroscopy, are elegant alternatives. A major disadvantage of Raman spectroscopy is the low signal yield resulting in long acquisition times, making it impractical for high‐throughput clinical analysis. As a rule, Raman‐based cell identification relies on high‐resolution Raman spectra. This comes at a cost of detected Raman photons. In this letter we show that while the proper biochemical characterization of cells requires high‐resolution Raman spectra, the proper classification of cells does not. By varying the slit‐width between 50 µm and 500 µm it is possible to show that detected Raman signal from eukaryotic cells increased up to seven‐fold. Raman‐based cell classification was performed on three cancer cell lines: Jurkat, MiaPaca2, and Capan1, at three different resolutions 8 cm^–1, 24 cm^–1, and 48 cm^–1. Moreover, we have simulated the resolution decrease due to low‐diffraction gratings by binning neighboring pixels together. In both cases the cells were well classifiable using support vectors machine (SVM).

For anyone working in the field of Raman spectroscopy this picture of Sir C.V. Raman is recognizable, even with reduced spatial resolution. Raman spectra of eukaryotic cells can also be recognized even with six fold reduced spectral resolution.     

Engineering tumors with 3D scaffolds

Microenvironmental conditions control tumorigenesis and biomimetic culture systems that allow for in vitro and in vivo tumor modeling may greatly aid studies of cancer cells' dependency on these conditions. We engineered three-dimensional (3D) human tumor models using carcinoma cells in polymeric scaffolds that recreated microenvironmental characteristics representative of tumors in vivo. Strikingly, the angiogenic characteristics of tumor cells were dramatically altered upon 3D culture within this system, and corresponded much more closely to tumors formed in vivo. Cells in this model were also less sensitive to chemotherapy and yielded tumors with enhanced malignant potential. We assessed the broad relevance of these findings with 3D culture of other tumor cell lines in this same model, comparison with standard 3D Matrigel culture and in vivo experiments. This new biomimetic model may provide a broadly applicable 3D culture system to study the effect of microenvironmental conditions on tumor malignancy in vitro and in vivo.     

Breast tumor cells with PI3K mutation or HER2 amplification are selectively addicted to Akt signaling

Background

Dysregulated PI3K/Akt signaling occurs commonly in breast cancers and is due to HER2 amplification, PI3K mutation or PTEN inactivation. The objective of this study was to determine the role of Akt activation in breast cancer as a function of mechanism of activation and whether inhibition of Akt signaling is a feasible approach to therapy.

Methodology/Principal Findings

A selective allosteric inhibitor of Akt kinase was used to interrogate a panel of breast cancer cell lines characterized for genetic lesions that activate PI3K/Akt signaling: HER2 amplification or PI3K or PTEN mutations in order to determine the biochemical and biologic consequences of inhibition of this pathway. A variety of molecular techniques and tissue culture and in vivo xenograft models revealed that tumors with mutational activation of Akt signaling were selectively dependent on the pathway. In sensitive cells, pathway inhibition resulted in D-cyclin loss, G1 arrest and induction of apoptosis, whereas cells without pathway activation were unaffected. Most importantly, the drug effectively inhibited Akt kinase and its downstream effectors in vivo and caused complete suppression of the growth of breast cancer xenografts with PI3K mutation or HER2 amplification, including models of the latter selected for resistance to Herceptin. Furthermore, chronic administration of the drug was well-tolerated, causing only transient hyperglycemia without gross toxicity to the host despite the pleiotropic normal functions of Akt.

Conclusions/Significance

These data demonstrate that breast cancers with PI3K mutation or HER2 amplification are selectively dependent on Akt signaling, and that effective inhibition of Akt in tumors is feasible and effective in vivo. These findings suggest that direct inhibition of Akt may represent a therapeutic strategy for breast and other cancers that are addicted to the pathway including tumors with resistant to Herceptin.     

LncRNA PCBP1-AS1-mediated AR/AR-V7 deubiquitination enhances prostate cancer enzalutamide resistance

The refractory of castration-resistant prostate cancer (CRPC) is mainly reflected in drug resistance. The current research on the resistance mechanism of CRPC is still in its infancy. In this study, we revealed for the first time the key role of LncRNA PCBP1-AS1 in CRPC drug resistance. Through detailed in vivo and in vitro studies, we found that PCBP1-AS1 may enhance the deubiquitination of AR/AR-V7 by stabilizing the USP22-AR/AR-V7 complex, thereby preventing AR/AR-V7 from being degraded through the ubiquitin–proteasome pathway. Targeting PCBP1-AS1 can significantly restore the drug sensitivity of enzalutamide-resistant tumors in vivo and in vitro. Our research further expands the function of LncRNA in castration-resistant prostate cancer, which may provide new potential for clinical diagnosis and targeted therapy.Subject terms: Prostate cancer, Ubiquitylation, Long non-coding RNAs     

Autofluorescence and white light imaging‐guided endoscopic Raman and diffuse reflectance spectroscopy for in vivo nasopharyngeal cancer detection

Nasopharyngeal cancer (NPC) is an endemic with high incidence in Southern China and Southeast Asia countries. Screening for NPC under conventional white light imaging (WLI) nasopharyngoscope examination remains a great clinical challenge due to its poor sensitivity. Here, we developed an integrated 4‐modality endoscopy system combining WLI, autofluorescence imaging (AFI), diffuse reflectance spectroscopy and Raman spectroscopy technologies for in vivo endoscopic cancer detection for the first time. A pilot clinical test of the system for NPC detection was conducted, in which 283 in vivo Raman and diffuse reflectance spectral data sets from 30 NPC patients and 30 healthy subjects were acquired under the guidance of AFI and WLI. Both high diagnostic sensitivity (98.6%) and high specificity (95.1%) for differentiating cancer from normal tissue sites were achieved using this system combined with principal component analysis‐linear discriminant analysis diagnostic algorithm, demonstrating great potential for improving real‐time, in vivo diagnosis of NPC at endoscopy.      

Portable spectroscopic system for in vivo skin neoplasms diagnostics by Raman and autofluorescence analysis

The present paper studies the applicability of a portable cost‐effective spectroscopic system for the optical screening of skin tumors. in vivo studies of Raman scattering and autofluorescence (AF) of skin tumors with the 785 nm excitation laser in the near‐infrared region included malignant melanoma, basal cell carcinoma and various types of benign neoplasms. The efficiency of the portable system was evaluated by comparison with a highly sensitive spectroscopic system and with the diagnosis accuracy of a human oncologist. Partial least square analysis of Raman and AF spectra was performed; specificity and sensitivity of various skin oncological pathologies detection varied from 78.9% to 100%. Hundred percent accuracy of benign and malignant skin tumors differentiation is possible only with a combined analysis of Raman and AF signals.      

Exploring the effect of vitamin E in cancer chemotherapy—A biochemical and biophysical insight

Many oncologists contend that patient undergoing chemotherapy must avoid antioxidant supplementation as it may interfere with the activity of the drug. In the present investigation, we have explored the influence of vitamin E, a well‐known antioxidant on Camptothecin (CPT), a potent anti‐cancer drug induced cell apoptosis and death of cervical cancer cells. HeLa cells were treated with different concentrations of CPT in presence and absence of 100 μm vitamin E. Treated cells were subjected to cytotoxicity studies, catalase assay, DNA fragmentation assay, clonogenic assay and flow cytometry based apoptosis detection. Also, Raman spectroscopy a label free technique which provides global information, in conjunction with multivariate tools like PCA, PCLDA and FDA, was investigated to explore vitamin E supplementation induced alterations. Our data based on biochemical and biophysical experimental analysis reveals that CPT causes DNA damage along with protein and lipid alteration culminating in cell death. Importantly, Raman spectroscopic analysis could uniquely differentiate the cluster of control and vitamin E control from CPT and CPT + Vit E treated cells. We conclusively prove that presence of vitamin E at 100 μM concentration shows promising antioxidant activity and displays no modulatory role on CPT induced effect, thereby causing no possible hindrance with the efficacy of the drug. Vitamin E may prove beneficial to alleviate chemotherapy associated side effects in patients during clinical settings which may open the doors further for subsequent exploration in in vivo preclinical studies.      

Fabrication techniques of biomimetic scaffolds in three‐dimensional cell culture: A review

In the last four decades, several researchers worldwide have routinely and meticulously exercised cell culture experiments in two‐dimensional (2D) platforms. Using traditionally existing 2D models, the therapeutic efficacy of drugs has been inappropriately validated due to the failure in generating the precise therapeutic response. Fortunately, a 3D model addresses the foregoing limitations by recapitulating the in vivo environment. In this context, one has to contemplate the design of an appropriate scaffold for favoring the organization of cell microenvironment. Instituting pertinent model on the platter will pave way for a precise mimicking of in vivo conditions. It is because animal cells in scaffolds oblige spontaneous formation of 3D colonies that molecularly, phenotypically, and histologically resemble the native environment. The 3D culture provides insight into the biochemical aspects of cell–cell communication, plasticity, cell division, cytoskeletal reorganization, signaling mechanisms, differentiation, and cell death. Focusing on these criteria, this paper discusses in detail, the diversification of polymeric scaffolds based on their available resources. The paper also reviews the well‐founded and latest techniques of scaffold fabrication, and their applications pertaining to tissue engineering, drug screening, and tumor model development.     

Microencapsulated multicellular tumor spheroids as a novel in vitro model for drug screening

To generate multicellular tumor spheroids (MTS) based on human breast adenocarcinoma MCF-7 cells and to study them as a novel in vitro model for anticancer drug screening, a technique for cell microencapsulation in biocompatible alginate-chitosan microcapsules has been used in this study. Using the MTS based on the MCF-7 cells methotrexate (MTX) cytotoxicity has been investigated. A set of MTS with an average size of 150, 200 and 300 μm was prepared as a function of cultivation time. Cell viability was evaluated after MTS incubation in cultivation medium containing various MTX concentrations (1, 2, 10, 50 and 100 nM) for 48 h. MTS were shown to be markedly more resistant to MTX than the monolayer culture. The increase of the spheroid size was in correlation with the enhanced MTS resistance to MTX. Thus, at 100 nM MTX a number of viable cells in MTS with the size of 300 μm was 2.5-fold higher than that in the monolayer culture. It is suggested that the cells microencapsulated into MTS can better mimic cell behavior in small solid tumors compared to the monolayer culture. In the future MTS could be proposed as a novel in vitro model for anticancer drug screening.     

Simultaneous fingerprint and high‐wavenumber fiber‐optic Raman spectroscopy enhances real‐time in vivo diagnosis of adenomatous polyps during colonoscopy

Colorectal cancer can be prevented if detected early (e.g., precancerous polyps‐adenoma). Endoscopic differential diagnosis of hyperplastic polyps (that have little or no risk of malignant transformation) and adenomas (that have prominent malignant latency) remains an unambiguous clinical challenge. Raman spectroscopy is an optical vibrational technique capable of probing biomolecular changes of tissue associated with neoplastic transformation. This work aims to apply a fiber‐optic simultaneous fingerprint (FP) and high wavenumber (HW) Raman spectroscopy technique for real‐time in vivo assessment of adenomatous polyps during clinical colonoscopy. We have developed a fiber‐optic Raman endoscopic technique capable of simultaneously acquiring both the FP (i.e., 800–1800 cm^–1) and HW (i.e., 2800–3600 cm^–1) Raman spectra from colorectal tissue subsurface (<200 µm) for real‐time assessment of colorectal carcinogenesis. In vivo FP/HW Raman spectra were acquired from 50 patients with 17 colorectal polyps during clinical colonoscopy. Prominent Raman spectral differences (p < 0.001) were found between hyperplastic (n = 118 spectra), adenoma (n = 184 spectra) that could be attributed to changes in inter‐ and intra‐cellular proteins, lipids, DNA and water structures and conformations. Simultaneous FP/HW Raman endoscopy provides a diagnostic sensitivity of 90.9% and specificity of 83.3% for differentiating adenoma from hyperplastic polyps, which is superior to either the FP or HW Raman technique alone. This study shows that simultaneous FP/HW Raman spectroscopy technique has the potential to be a clinically powerful tool for improving early diagnosis of adenomatous polyps in vivo during colonoscopic examination.

     

AlgiMatrix? Based 3D Cell Culture System as an In-Vitro Tumor Model for Anticancer Studies

Background

Three-dimensional (3D) in-vitro cultures are recognized for recapitulating the physiological microenvironment and exhibiting high concordance with in-vivo conditions. Taking the advantages of 3D culture, we have developed the in-vitro tumor model for anticancer drug screening.

Methods

Cancer cells grown in 6 and 96 well AlgiMatrix™ scaffolds resulted in the formation of multicellular spheroids in the size range of 100–300 µm. Spheroids were grown in two weeks in cultures without compromising the growth characteristics. Different marketed anticancer drugs were screened by incubating them for 24 h at 7, 9 and 11 days in 3D cultures and cytotoxicity was measured by AlamarBlue® assay. Effectiveness of anticancer drug treatments were measured based on spheroid number and size distribution. Evaluation of apoptotic and anti-apoptotic markers was done by immunohistochemistry and RT-PCR. The 3D results were compared with the conventional 2D monolayer cultures. Cellular uptake studies for drug (Doxorubicin) and nanoparticle (NLC) were done using spheroids.

Results

IC₅₀ values for anticancer drugs were significantly higher in AlgiMatrix™ systems compared to 2D culture models. The cleaved caspase-3 expression was significantly decreased (2.09 and 2.47 folds respectively for 5-Fluorouracil and Camptothecin) in H460 spheroid cultures compared to 2D culture system. The cytotoxicity, spheroid size distribution, immunohistochemistry, RT-PCR and nanoparticle penetration data suggested that in vitro tumor models show higher resistance to anticancer drugs and supporting the fact that 3D culture is a better model for the cytotoxic evaluation of anticancer drugs in vitro.

Conclusion

The results from our studies are useful to develop a high throughput in vitro tumor model to study the effect of various anticancer agents and various molecular pathways affected by the anticancer drugs and formulations.     

Evaluation of cellular adhesion and organization in different microporous polymeric scaffolds

The lack of prediction accuracy during drug development and screening risks complications during human trials, such as drug‐induced liver injury (DILI), and has led to a demand for robust, human cell‐based, in vitro assays for drug discovery. Microporous polymer‐based scaffolds offer an alternative to the gold standard flat tissue culture plastic (2D TCPS) and other 3D cell culture platforms as the porous material entraps cells, making it advantageous for automated liquid handlers and high‐throughput screening (HTS). In this study, we optimized the surface treatment, pore size, and choice of scaffold material with respect to cellular adhesion, tissue organization, and expression of complex physiologically relevant (CPR) outcomes such as the presence of bile canaliculi‐like structures. Poly‐l‐ lysine and fibronectin (FN) coatings have been shown to encourage cell attachment to the underlying substrate. Treatment of the scaffold surface with NaOH followed with a coating of FN improved cell attachment and penetration into pores. Of the two pore sizes we investigated (A: 104 ± 4 μm; B: 175 ± 6 μm), the larger pore size better promoted cell penetration while limiting tissue growth from reaching the hypoxia threshold. Finally, polystyrene (PS) proved to be conducive to cell growth, penetration into the scaffold, and yielded CPR outcomes while being a cost‐effective choice for HTS applications. These observations provide a foundation for optimizing microporous polymer‐based scaffolds suitable for drug discovery. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:505–514, 2018     

Enhancing the evaluation of PI3K inhibitors through 3D melanoma models

Targeted therapies for mutant BRAF metastatic melanoma are effective but not curative due to acquisition of resistance. PI3K signaling is a common mediator of therapy resistance in melanoma; thus, the need for effective PI3K inhibitors is critical. However, testing PI3K inhibitors in adherent cultures is not always reflective of their potential in vivo. To emphasize this, we compared PI3K inhibitors of different specificity in two‐ and three‐dimensional (2D, 3D) melanoma models and show that drug response predictions gain from evaluation using 3D models. Our results in 3D demonstrate the anti‐invasive potential of PI3K inhibitors and that drugs such as PX‐866 have beneficial activity in physiological models alone and when combined with BRAF inhibition. These assays finally help highlight pathway effectors that could be involved in drug response in different environments (e.g. p4E‐BP1). Our findings show the advantages of 3D melanoma models to enhance our understanding of PI3K inhibitors.