Quantitative analysis of tumour spheroid structure.

Tumour spheroids are common in vitro experimental models of avascular tumour growth. Compared with traditional two-dimensional culture, tumour spheroids more closely mimic the avascular tumour microenvironment where spatial differences in nutrient availability strongly influence growth. We show that spheroids initiated using significantly different numbers of cells grow to similar limiting sizes, suggesting that avascular tumours have a limiting structure; in agreement with untested predictions of classical mathematical models of tumour spheroids. We develop a novel mathematical and statistical framework to study the structure of tumour spheroids seeded from cells transduced with fluorescent cell cycle indicators, enabling us to discriminate between arrested and cycling cells and identify an arrested region. Our analysis shows that transient spheroid structure is independent of initial spheroid size, and the limiting structure can be independent of seeding density. Standard experimental protocols compare spheroid size as a function of time; however, our analysis suggests that comparing spheroid structure as a function of overall size produces results that are relatively insensitive to variability in spheroid size. Our experimental observations are made using two melanoma cell lines, but our modelling framework applies across a wide range of spheroid culture conditions and cell lines.

An in vitro tumorigenesis model based on live-cell-generated oxygen and nutrient gradients.

The tumor microenvironment (TME) is multi-cellular, spatially heterogenous, and contains cell-generated gradients of soluble molecules. Current cell-based model systems lack this complexity or are difficult to interrogate microscopically. We present a 2D live-cell chamber that approximates the TME and demonstrate that breast cancer cells and macrophages generate hypoxic and nutrient gradients, self-organize, and have spatially varying phenotypes along the gradients, leading to new insights into tumorigenesis.

Understanding the influence of substrate when growing tumorspheres.

BACKGROUND: Cancer stem cells are important for the development of many solid tumors. These cells receive promoting and inhibitory signals that depend on the nature of their environment (their niche) and determine cell dynamics. Mechanical stresses are crucial to the initiation and interpretation of these signals. METHODS: A two-population mathematical model of tumorsphere growth is used to interpret the results of a series of experiments recently carried out in Tianjin, China, and extract information about the intraspecific and interspecific interactions between cancer stem cell and differentiated cancer cell populations. RESULTS: The model allows us to reconstruct the time evolution of the cancer stem cell fraction, which was not directly measured. We find that, in the presence of stem cell growth factors, the interspecific cooperation between cancer stem cells and differentiated cancer cells induces a positive feedback loop that determines growth, independently of substrate hardness. In a frustrated attempt to reconstitute the stem cell niche, the number of cancer stem cells increases continuously with a reproduction rate that is enhanced by a hard substrate. For growth on soft agar, intraspecific interactions are always inhibitory, but on hard agar the interactions between stem cells are collaborative while those between differentiated cells are strongly inhibitory. Evidence also suggests that a hard substrate brings about a large fraction of asymmetric stem cell divisions. In the absence of stem cell growth factors, the barrier to differentiation is broken and overall growth is faster, even if the stem cell number is conserved. CONCLUSIONS: Our interpretation of the experimental results validates the centrality of the concept of stem cell niche when tumor growth is fueled by cancer stem cells. Niche memory is found to be responsible for the characteristic population dynamics observed in tumorspheres. The model also shows why substratum stiffness has a deep influence on the behavior of cancer stem cells, stiffer substrates leading to a larger proportion of asymmetric doublings. A specific condition for the growth of the cancer stem cell number is also obtained.

Guided assembly of cancer ellipsoid on suspended hydrogel microfibers estimates multi-cellular traction force.

Three-dimensional (3D) multi-cellular aggregates hold important applications in tissue engineering and in vitro biological modeling. Probing the intrinsic forces generated during the aggregation process, could open up new possibilities in advancing the discovery of tissue mechanics-based biomarkers. We use individually suspended, and tethered gelatin hydrogel microfibers to guide multicellular aggregation of brain cancer cells (glioblastoma cell line, U87), forming characteristic cancer 'ellipsoids'. Over a culture period of up to 13 days, U87 aggregates evolve from a flexible cell string with cell coverage following the relaxed and curly fiber contour; to a distinct ellipsoid-on-string morphology, where the fiber segment connecting the ellipsoid poles become taut. Fluorescence imaging revealed the fiber segment embedded within the ellipsoidal aggregate to exhibit a morphological transition analogous to filament buckling under a compressive force. By treating the multicellular aggregate as an effective elastic medium where the microfiber is embedded, we applied a filament post-buckling theory to model the fiber morphology, deducing the apparent elasticity of the cancer ellipsoid medium, as well as the collective traction force inherent in the aggregation process.

Mathematical modelling reveals cellular dynamics within tumour spheroids.

Tumour spheroids are widely used as an in vitro assay for characterising the dynamics and response to treatment of different cancer cell lines. Their popularity is largely due to the reproducible manner in which spheroids grow: the diffusion of nutrients and oxygen from the surrounding culture medium, and their consumption by tumour cells, causes proliferation to be localised at the spheroid boundary. As the spheroid grows, cells at the spheroid centre may become hypoxic and die, forming a necrotic core. The pressure created by the localisation of tumour cell proliferation and death generates an cellular flow of tumour cells from the spheroid rim towards its core. Experiments by Dorie et al. showed that this flow causes inert microspheres to infiltrate into tumour spheroids via advection from the spheroid surface, by adding microbeads to the surface of tumour spheroids and observing the distribution over time. We use an off-lattice hybrid agent-based model to re-assess these experiments and establish the extent to which the spatio-temporal data generated by microspheres can be used to infer kinetic parameters associated with the tumour spheroids that they infiltrate. Variation in these parameters, such as the rate of tumour cell proliferation or sensitivity to hypoxia, can produce spheroids with similar bulk growth dynamics but differing internal compositions (the proportion of the tumour which is proliferating, hypoxic/quiescent and necrotic/nutrient-deficient). We use this model to show that the types of experiment conducted by Dorie et al. could be used to infer spheroid composition and parameters associated with tumour cell lines such as their sensitivity to hypoxia or average rate of proliferation, and note that these observations cannot be conducted within previous continuum models of microbead infiltration into tumour spheroids as they rely on resolving the trajectories of individual microbeads.

Axl-148b chimeric aptamers inhibit breast cancer and melanoma progression.

microRNAs (miRNAs) are small non-coding RNAs acting as negative regulators of gene expression and involved in tumor progression. We recently showed that inhibition of the pro-metastatic miR-214 and simultaneous overexpression of its downstream player, the anti-metastatic miR-148b, strongly reduced metastasis formation. To explore the therapeutic potential of miR-148b, we generated a conjugated molecule aimed to target miR-148b expression selectively to tumor cells. Precisely, we linked miR-148b to GL21.T, an aptamer able to specifically bind to AXL, an oncogenic tyrosine kinase receptor highly expressed on cancer cells. Axl-148b conjugate was able to inhibit migration and invasion of AXL-positive, but not AXL-negative, cancer cells, demonstrating high efficacy and selectivity in vitro. In parallel, expression of ALCAM and ITGA5, two miR-148b direct targets, was reduced. More importantly, axl-148b chimeric aptamers were able to inhibit formation and growth of 3D-mammospheres, to induce necrosis and apoptosis of treated xenotransplants, as well as to block breast cancer and melanoma dissemination and metastatization in mice. Relevantly, axl aptamer acted as specific delivery tool for miR-148b, but it also actively contributed to inhibit metastasis formation, together with miR-148b. In conclusion, our data show that axl-148b conjugate is able to inhibit tumor progression in an axl- and miR-148b-dependent manner, suggesting its potential development as therapeutic molecule.

Quantitative cell-based model predicts mechanical stress response of growing tumor spheroids over various growth conditions and cell lines.

Model simulations indicate that the response of growing cell populations on mechanical stress follows the same functional relationship and is predictable over different cell lines and growth conditions despite experimental response curves look largely different. We develop a hybrid model strategy in which cells are represented by coarse-grained individual units calibrated with a high resolution cell model and parameterized by measurable biophysical and cell-biological parameters. Cell cycle progression in our model is controlled by volumetric strain, the latter being derived from a bio-mechanical relation between applied pressure and cell compressibility. After parameter calibration from experiments with mouse colon carcinoma cells growing against the resistance of an elastic alginate capsule, the model adequately predicts the growth curve in i) soft and rigid capsules, ii) in different experimental conditions where the mechanical stress is generated by osmosis via a high molecular weight dextran solution, and iii) for other cell types with different growth kinetics from the growth kinetics in absence of external stress. Our model simulation results suggest a generic, even quantitatively same, growth response of cell populations upon externally applied mechanical stress, as it can be quantitatively predicted using the same growth progression function.

Effects of human umbilical cord mesenchymal stem cells on co-cultured ovarian carcinoma cells.

Ovarian carcinoma is mainly treated by surgery aided by chemotherapy. If supplemented by stem cells treatment, its recurrence rate and mortality rate will be decreased. This is a new therapy. In this study, ovarian cancer cells were cultured together with umbilical cord mesenchymal stem cells (UCMSCs), and the interactions between them were observed. The results showed that the survival rates of UCMSCs increased to 83.8 ± 2.2% from 56.5 ± 5.5%, and the survival rates of ovarian cancer cells decreased to 16.2 ± 2.2% from 43.5 ± 5.5% with the progression of the cultural time from 24 to 96 hr. There was a significant difference between them (p < .05). It revealed that UCMSCs could inhibit the proliferation of ovarian cancer cells.

Aberrant Super-Enhancer Landscape in Human Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) cells exploit an aberrant transcriptional program to sustain their infinite growth and progression. Emerging evidence indicates that the continuous and robust transcription of oncogenes in cancer cells is often driven by super-enhancers (SEs). In this study, we systematically compared the SE landscapes between normal liver and HCC cells and revealed that the cis-acting SE landscape was extensively reprogrammed during liver carcinogenesis. HCC cells acquired SEs at multiple prominent oncogenes to drive their vigorous expression. We identified sphingosine kinase 1 (SPHK1) as an SE-associated oncogene, and we used this gene as an example to illustrate the impact of SEs on the activation of oncogenes in HCC. Concurrently, we also showed that the critical components of the trans-acting SE complex, namely, cyclin-dependent kinase 7 (CDK7), bromodomain-containing protein 4 (BRD4), E1A binding protein P300 (EP300), and mediator complex subunit 1 (MED1), were frequently overexpressed in human HCCs and were associated with the poor prognosis of patients with HCC. Using the CRISPR/Cas9 gene-editing system and specific small-molecule inhibitors, we further demonstrated that HCC cells were highly sensitive to perturbations of the SE complex. The inactivation of CDK7, BRD4, EP300, and MED1 selectively repressed the expression of SE-associated oncogenes in HCC. Finally, we demonstrated that THZ1, which is a small-molecule inhibitor of CDK7, exerted a prominent anticancer effect in both in vitro and in vivo HCC models. Conclusion: The SE landscape and machinery were significantly altered in human HCCs. HCC cells are highly susceptible to perturbations of the SE complex due to the resulting selective suppression of SE-associated oncogenes. Our results suggest that targeting SE complex is a promising therapeutic strategy for HCC treatment.

Studying Cell Mechanobiology in 3D: The Two-Photon Lithography Approach.

Two-photon lithography is a laser writing technique that can produce 3D microstructures with resolutions below the diffraction limit. This review focuses on its applications to study mechanical properties of cells, an emerging field known as mechanobiology. We review 3D structural designs and materials in the context of new experimental designs, including estimating forces exerted by single cells, studying selective adhesion on substrates, and creating 3D networks of cells. We then focus on emerging applications, including structures for assessing cancer cell invasiveness, whose migration properties depend on the cell mechanical response to the environment, and 3D architectures and materials to study stem cell differentiation, as 3D structure shape and patterning play a key role in defining cell fates.

Experimental estimation of stored stress within spherical microtissues : What can and cannot be inferred from cutting experiments.

Biological tissues accumulate mechanical stress during their growth. The mere measurement of the stored stress is not an easy task. We address here the spherical case and our experiments consist in performing an incision of a spherical microtissue (tumor spheroid) grown in vitro. On the theoretical part we derive a compatibility condition on the stored stress in spherical symmetry, which imposes a relation between the circumferential and radial stored stress. The numerical implementation uses the hyperelastic model of Ciarlet and Geymonat. A parametric study is performed to assess the influence of each parameter on the shape of the domain after the incision. As a conclusion, the total radial stored stress can be confidently estimated from the measurement of the opening after incision. We validate the approach with experimental data.

Colorectal cancer spheroid biobanks: multi-level approaches to drug sensitivity studies.

Biobanking of molecularly characterized colorectal cancer stem cells (CSCs) generated from individual patients and growing as spheroids in defined serum-free media offer a fast, feasible, and multi-level approach for the screening of targeted therapies and drug resistance molecular studies. By combining in vitro and in vivo analyses of cetuximab efficacy with genetic data on an ongoing collection of stem cell-enriched spheroids, we describe the identification and preliminary characterization of microsatellite stable (MSS) CSCs that, despite the presence of the KRAS (G12D) mutation, display epidermal growth factor (EGF)-dependent growth and are strongly inhibited by anti-EGF-receptor (EGFR) treatment. In parallel, we detected an increased resistance to anti-EGFR therapy of microsatellite instable (MSI) CSC lines irrespective of KRAS mutational status. MSI CSC lines carried mutations in genes coding for proteins with a role in RAS and calcium signaling, highlighting the role of a genomically unstable context in determining anti-EGFR resistance. Altogether, these results argue for a multifactorial origin of anti-EGFR resistance that emerges as the effect of multiple events targeting direct and indirect regulators of the EGFR pathway. An improved understanding of key molecular determinants of sensitivity/resistance to EGFR inhibition will be instrumental to optimize the clinical efficacy of anti-EGFR agents, representing a further step towards personalized treatments.

Rabdocoestin B exhibits antitumor activity by inducing G2/M phase arrest and apoptosis in esophageal squamous cell carcinoma.

PURPOSE: Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive squamous cell carcinomas and is generally resistant to chemotherapy. In the present study, the cytotoxic activity of Rabdocoestin B (Rabd-B) against ESCC and the underlying mechanisms were investigated. METHODS: The inhibitory effect of Rabd-B on KYSE30 and KYSE450 was evaluated by Cell Counting Kit-8 (CCK8) and colony formation assays in vitro. The cell cycle distribution and apoptosis of cells treated with Rabd-B were determined by flow cytometry. The mechanisms underlying the effects of Rabd-B were systematically examined by Western blot. The in vivo anti-tumor ability of Rabd-B was measured in mouse xenograft models and cisplatin (DDP) was used as positive control. RESULTS: Rabd-B efficiently induced G2/M phase arrest in ESCC cells by upregulating the Chk1/Chk2-Cdc25C axis to inhibit the G2→M transition facilitated by Cdc2/Cyclin B1. Furthermore, Rabd-B suppressed ATM/ATR phosphorylation, thereby inhibiting BRCA1-mediated DNA repair, which resulted in mitotic catastrophe and induced cell apoptosis. Rabd-B also decreased the activity of the Akt and NF-κB survival signaling pathways and ultimately initiated the caspase-9-dependent intrinsic apoptotic pathway in ESCC cells. The apoptosis induced by Rabd-B could be partially reversed by a caspase-9-specific inhibitor (Z-LEHD-FMK) and a pan-caspase inhibitor (Z-VAD-FMK). Moreover, Rabd-B effectively suppressed tumor growth in mouse xenografts which was comparable to that of DDP without significant injuries to the mice. CONCLUSION: Taken together, these findings indicate that Rabd-B is a promising precursor compound that may be useful as a treatment for ESCC and thus warrants further investigation.

Kaposi's Sarcoma-Associated Herpesvirus Infection Modulates the Proliferation of Glioma Stem-Like Cells.

Glioblastoma multiforme is the most lethal malignant brain tumor. Despite many intensive studies, the prognosis of glioblastoma multiforme is currently very poor, with a median overall survival duration of 14 months and 2-year survival rates of less than 10%. Although viral infections have been emphasized as potential cofactors, their influences on pathways that support glioblastoma progression are not known. Some previous studies indicated that human Kaposi's sarcoma-associated herpesvirus (KSHV) was detected in healthy brains, and its microRNA was also detected in glioblastoma patients' plasma. However, a direct link between KSHV infection and glioblastoma is currently not known. In this study, we infected glioblastoma cells and glioma stem-like cells (GSCs) with KSHV to establish an in vitro cell model for KSHV-infected glioblastoma cells and glioma stem-like cells in order to identify virologic outcomes that overlap with markers of aggressive disease. Latently KSHV-infected glioblastoma cells and GSCs were successfully established. Additionally, using these cell models, we found that KSHV infection modulates the proliferation of glioma stem-like cells.

AFM-detected apoptosis of hepatocellular carcinoma cells induced by American ginseng root water extract.

American ginseng as a common and traditional herbal medicine has been used in cancer treatment for many years. However, the effect of American ginseng on the cancer cell response (i.e. apoptosis) has not been fully understood yet. Previous studies demonstrated that cellular apoptosis was associated with the changes of mechanical and morphological properties. Therefore, in this study, mechanical and morphological characterizations were carried out by both atomic force microscope (AFM) and inverted optical microscope to investigate the apoptosis of hepatocellular carcinoma (SMMC-7721) cells affected by American ginseng root water extract (AGRWE). The results showed that the cells treated with AGRWE exhibited significantly larger surface roughness, height and elastic modulus values than control group. Moreover, those parameters were upregulated under the higher concentration of AGRWE and longer culture time. Consequently, it indicates that the mechanical and morphological properties can be used as the apoptotic characteristics of SMMC-7721 cells. Also, the increased surface roughness and elastic modulus of cells under the AGRWE treatment have shown that the apoptosis of SMMC-7721 cells can be enhanced by AGRWE. This will provide an important implication for hepatocelluar carcinoma treatment and drug development.

KCa3.1 Channels and Glioblastoma: In Vitro Studies.

BACKGROUND: Several tumor entities including brain tumors aberrantly overexpress intermediate conductance Ca2+ activated KCa3.1 K+ channels. These channels contribute significantly to the transformed phenotype of the tumor cells. METHOD: PubMed was searched in order to summarize our current knowledge on the molecular signaling upstream and downstream and the effector functions of KCa3.1 channel activity in tumor cells in general and in glioblastoma cells in particular. In addition, KCa3.1 expression and function for repair of DNA double strand breaks was determined experimentally in primary glioblastoma cultures in dependence on the abundance of proneural and mesenchymal stem cell markers. RESULTS: By modulating membrane potential, cell volume, Ca2+ signals and the respiratory chain, KCa3.1 channels in both, plasma and inner mitochondrial membrane, have been demonstrated to regulate many cellular processes such as migration and tissue invasion, metastasis, cell cycle progression, oxygen consumption and metabolism, DNA damage response and cell death of cancer cells. Moreover, KCa3.1 channels have been shown to crucially contribute to resistance against radiotherapy. Futhermore, the original in vitro data on KCa3.1 channel expression in subtypes of glioblastoma stem(-like) cells propose KCa3.1 as marker for the mesenchymal subgroup of cancer stem cells and suggest that KCa3.1 contributes to the therapy resistance of mesenchymal glioblastoma stem cells. CONCLUSION: The data suggest KCa3.1 channel targeting in combination with radiotherapy as promising new tool to eradicate therapy-resistant mesenchymal glioblastoma stem cells.

Non-malignant epithelial cells preferentially proliferate from nasopharyngeal carcinoma biopsy cultured under conditionally reprogrammed conditions.

Nasopharyngeal carcinoma (NPC) is an invasive cancer with particularly high incidence in Southern China and Southeast Asia. The study of NPC is greatly hampered by the lack of reliable cell lines due to the loss of EBV genome and HeLa cell contamination. Conditional reprogramming (CR) cell culture technique has been reported for rapid and efficient establishment of patient-derived normal and tumor cell cultures. The purpose of this study was to assess this method to culture NPC patient-derived primary tumor cells. Using CR protocol, we demonstrated that epithelial cells could be efficiently cultured from normal (70%) and cancerous nasopharyngeal (46%) biopsies. However, by comparing with original tumors in terms of mutation and methylation profiles, epithelial cells derived from cancerous biopsy represented non-malignant cells. Further, they exhibited stem-like characteristics based on their cell surface proteins and could differentiate into pseudostratified epithelium in an air-liquid interface culture system. We conclude that CR method is a highly selective and useful method for growing non-malignant nasopharyngeal epithelial cells.

Retinoblastoma protein (Rb) links hypoxia to altered mechanical properties in cancer cells as measured by an optical tweezer.

Hypoxia modulates actin organization via multiple pathways. Analyzing the effect of hypoxia on the biophysical properties of cancer cells is beneficial for studying modulatory signalling pathways by quantifying cytoskeleton rearrangements. We have characterized the biophysical properties of human LNCaP prostate cancer cells that occur in response to loss of the retinoblastoma protein (Rb) under hypoxic stress using an oscillating optical tweezer. Hypoxia and Rb-loss increased cell stiffness in a fashion that was dependent on activation of the extracellular signal-regulated kinase (ERK) and the protein kinase B (AKT)- mammalian target of rapamycin (MTOR) pathways. Pharmacological inhibition of MEK1/2, AKT or MTOR impeded hypoxia-inducible changes in the actin cytoskeleton and inhibited cell migration in Rb-deficient cells conditioned with hypoxia. These results suggest that loss of Rb in transformed hypoxic cancer cells affects MEK1/2-ERK/AKT-MTOR signalling and promotes motility. Thus, the mechanical characterization of cancer cells using an optical tweezer provides an additional technique for cancer diagnosis/prognosis and evaluating therapeutic performance.

PSMA-Targeted Theranostic Nanocarrier for Prostate Cancer.

Herein, we report the use of a theranostic nanocarrier (Folate-HBPE(CT20p)) to deliver a therapeutic peptide to prostate cancer tumors that express PSMA (folate hydrolase 1). The therapeutic peptide (CT20p) targets and inhibits the chaperonin-containing TCP-1 (CCT) protein-folding complex, is selectively cytotoxic to cancer cells, and is non-toxic to normal tissue. With the delivery of CT20p to prostate cancer cells via PSMA, a dual level of cancer specificity is achieved: (1) selective targeting to PSMA-expressing prostate tumors, and (2) specific cytotoxicity to cancer cells with minimal toxicity to normal cells. The PSMA-targeting theranostic nanocarrier can image PSMA-expressing cells and tumors when a near infrared dye is used as cargo. Meanwhile, it can be used to treat PSMA-expressing tumors when a therapeutic, such as the CT20p peptide, is encapsulated within the nanocarrier. Even when these PSMA-targeting nanocarriers are taken up by macrophages, minimal cell death is observed in these cells, in contrast with doxorubicin-based therapeutics that result in significant macrophage death. Incubation of PSMA-expressing prostate cancer cells with the Folate-HBPE(CT20p) nanocarriers induces considerable changes in cell morphology, reduction in the levels of integrin ß1, and lower cell adhesion, eventually resulting in cell death. These results are relevant as integrin ß1 plays a key role in prostate cancer invasion and metastatic potential. In addition, the use of the developed PSMA-targeting nanocarrier facilitates the selective in vivo delivery of CT20p to PSMA-positive tumor, inducing significant reduction in tumor size.

Rapid and Short-term Extracellular Matrix-mediated In Vitro Culturing of Tumor and Nontumor Human Primary Prostate Cells From Fresh Radical Prostatectomy Tissue.

OBJECTIVE: To culture prostate cells from fresh biopsy core samples from radical prostatectomy (RP) tissue. Further, given the genetic heterogeneity of prostate cells, the ability to culture single cells from primary prostate tissue may be of importance toward enabling single-cell characterization of primary prostate tissue via molecular and cellular phenotypic biomarkers. METHODS: A total of 260 consecutive tissue samples from RPs were collected between October 2014 and January 2016, transported at 4°C in serum-free media to an off-site central laboratory, dissociated, and cultured. A culture protocol, including a proprietary extracellular matrix formulation (ECMf), was developed that supports rapid and short-term single-cell culture of primary human prostate cells derived from fresh RP samples. RESULTS: A total of 251 samples, derived from RP samples, yielded primary human tumor and nontumor prostate cells. Cultured cells on ECMf exhibit (1) survival after transport from the operating room to the off-site centralized laboratory, (2) robust (>80%) adhesion and survival, and (3) expression of different cell-type-specific markers. Cells derived from samples of increasing Gleason score exhibited a greater number of focal adhesions and more focal adhesion activation as measured by phospho-focal adhesion kinase (Y397) immunofluorescence when patient-derived cells were cultured on ECMf. Increased Ki67 immunofluorescence levels were observed in cells derived from cancerous RP tissue when compared to noncancerous RP tissue. CONCLUSION: By utilizing a unique and defined extracellular matrix protein formulation, tumor and nontumor cells derived from primary human prostate tissue can be rapidly cultured and analyzed within 72 hours after harvesting from RP tissue.