To further confirm the data and generate quantitative measures, the number of living cells inside VITVO for each cell line was then evaluated using the Real Time-Glo MT cell viability assay with a luminometer, as an additional applicable read-out in combination with the microscopy. The addition of Real Time-Glo reagent in culture media produce a bioluminescence signal proportional to the number of viable cells. Cells loaded in VITVO and incubated with Real Time-Glo generated a gradual increase in bioluminescence signal, thus indicating viable cells progressively proliferate and colonize the 3D matrix Fig.
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Using this assay, cell growth could be directly monitored over time inside the system by measuring the relative light units RLU. Considering the number of cells 5. These findings indicate VITVO is biocompatible and can sustain a 3D cell growth in a variety of cancer types which can be monitored using both microscopy and luminometry. After observing the capacity of VITVO to provide a 3D support for cancer cell line proliferation, the use of the system as a tool to monitor the impact of a known chemotherapy agent against a selected cell line was investigated.
Tumor cell viability was regularly monitored using both microscopy and Real Time-Glo assay.
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Untreated cells were used as control. In addition, decoy receptors can interfere with the mechanism of action Moreover, sTRAIL has a relevant molecular weight at least 36 KDa significantly higher than a chemical compound, possibly interfering with its biodistribution and penetration in a 3D structure. Thus, VITVO can be used to explore the anti-cancer potential of biologic agents in 3D and the addition of luciferase in target cells is suitable for comparative studies that may also involve parallel in vivo investigations.
Based on the results obtained using VITVO against tumor cell lines, whether the system could also host human primary tumor cells together with their accessory cells was investigate, as prerequisite before introducing the device as a predictive tool in oncology. Specifically, in addition to histological assessment of PD1-PDL1, the focus was on immuno-oncology agents, since rapid functional tests that can predict the efficacy of checkpoint inhibitors are lacking As shown in Fig.
Untreated samples were used as control. Representative images of untreated sample left pic and treated sample right pic are shown. In addition to the Real Time-Glo to visualize tumor cell death in the matrix using microscopy, another assay that can discriminate between living green and dying cells red; Fig. The progressive introduction of cancer cell therapies into the clinic, in addition to animal models that may be limited in hosting human cancers 18 , should require in vitro tools that can mimic the clinical setting in which cell growth can influence molecular biomarkers and antigenic profiles 19 , 20 , 21 , Therefore, whether a 3D system such as VITVO may have value in simultaneously hosting target and effector cells and identifying the effective dose compared with the dose determined in an animal study was investigated.
Then, luciferin was added to the system to evaluate cell viability at different time points by measuring RLU. Untreated mice were used as control. Tumor mass growth was monitored during the experiment, and after 28 days, a statistically significative reduction in tumor volume compared with untreated group was observed only in the E:T group.
Cell culture models are fundamental instruments for basic to translational research, in particular for cancer 24 , Although in vitro conditions do not exactly replicate in vivo situations, their role remains important 3. Conversely, while in vivo data are critical in drug discovery, a comprehensive survey of drug screening tests has shown that approximately half of toxicological data derived from experiments with rodents does not correlate with the results from human trials Moreover, pre-clinical toxicity studies with multiple animal species may be poorly predictive, and in several cases, drugs have failed during clinical trials in humans due to unexpected adverse toxicities 27 , Therefore, development of new in vitro models that can mimic the in vivo complexity and in combination with reliable animal models are needed to overcome these issues.
Based on bioreactor technology and considering these unmet needs, we developed VITVO as an innovative 3D tool providing a tissue-sized platform where cells can recreate their environment with a higher level of complexity and in a simple manner. Focusing on experimental oncology, the VITVO flexibility allowed testing of several approaches with a variety of read-outs.
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Conventional assays developed to study biological function and for testing anti-tumor pharmacodynamics are generally performed using 2D monolayer cultures; however, limits in predictability of the in vivo outcomes are well documented 1 , 2. Therefore, 3D cancer cell cultures are expected to better mimic in vivo microenvironments, providing an improved understanding of their morphological and functional peculiarities 21 , The miniaturization of both 2D and 3D cultures facilitated high throughput screening HTS technology, allowing the possibility to screen a large number of compounds evaluating the efficacy of a single endpoint 9.
However, this approach may not be completely predictive of the compound response in vivo , thus generating a high attrition rate in downstream drug development pipelines The gap between in vitro and in vivo may be bridged by creating more reliable assays for HTS or conceiving breakthrough technology based on low throughput screening LTS that can generate high value data with an increased level of prediction.
This situation may represent a limitation that often clashes with the possibility to obtain uniform structures able to mimic complexity and ensure reproducibility VITVO is a valuable tool to host a higher number of cells recreating a critical tumor mass with the possibility to generate an increased level of complexity by co-culturing different cell types and better mimic an in vivo microenvironment within a defined morphometry due to the shape of the inner core of the 3D system. As reported for other 3D cultures on scaffold 32 , 33 , VITVO is limited in the direct measurement of tumor size reduction after treatment, however it assesses cell viability in comparison to controls similarly to what is done when luciferase assay is performed in vivo.
Tumor Chemoresistance Assays
In that case, even if tumor size and residual cell number are not directly measured, the number of photons correlates with the reduction of tumor size and viable cells. Being aware of this limitation and in the attempt to overcome it, VITVO can be processed for histology where tumor mass can be also visualized for more direct histomorphological measurements Supplementary Fig. Therefore, the system can be used in LTS strategies and the platform was validated using different types of cancer cells including mesenchymal, epithelial, and neuroectodermal tumors.
Based on the data, the device can host various cell types that can be loaded into the device, repopulate the matrix, and survive in the closed environment due to the gas permeable membranes. In addition, the possibility to load a mix of different cells types originating either from freshly digested tumor samples or established cells lines i. MSC and tumors was demonstrated. Consequently, potential applications in which VITVO can be used to study the interactions between tumors and the different cells in their microenvironments lymphocyte-subsets, myeloid derived cells, stroma, and endothelium , without limiting the number and type of cells, can be developed.
Importantly, the flat nature of the system allows the monitoring of 3D matrix colonization and interaction due to cell labeling visible using microscopy and plate readers utilizing both fluorescence and luminescence. Finally, the structure enables the specific cancer niches to remain stable in the 3D matrix, allowing to follow them using microscopy without the risk of movement that could impair identification during the experimental period. All these factors are relevant for better understanding the dynamic interactions of a tumor and rebuilding its environment in VITVO for metabolic, immuno-oncology and basic genomic investigations.
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The simplicity of use and the adaptability to the common laboratory equipment may allow a rapid transfer of the technology into both academic and pharmaceutical research and development fields. Due to the closed system design, VITVO additionally offers the advantage of transportability; the tumor sample can be loaded at the clinical facility and then shipped to a remote laboratory for the necessary tests. An additional relevant application of the VITVO system is in the field of immuno-oncology having value both in drug development and clinical settings, where rapid functional tests allowing prediction of patient response are still lacking 9.
In the present study, freshly isolated cells collected from lung cancer patients and loaded in VITVO without the addition of effector cells, elicited the TIL activation caused by nivolumab against tumor cells. To the best of our knowledge, this process has not been previously described and presumably, the 3D growth generates favorable cell-to-cell interactions playing a key role in obtaining a closer relationship between target and effectors, as observed in patients.
Subsequently, an anti-tumor response is generated due to anti-PD1 antibodies and for better tumor antigen expression caused by 3D cell growth, as previously reported 21 , In conclusion, a novel device for 3D cell culture was introduced in the present study.
Cancer Immunotherapy Models
The simplicity of use and readout flexibility of VITVO allows a variety of pre-clinical investigations in oncology, with a possible relevant impact in other areas, such as toxicology studies using hepatocytes and for other more complex toxicology assays using inducible progenitors. Next, 5. This protocol was designed to ensure sufficient quantity of reagent considering the enlarged model compared with the miniaturized system.
The reagent was re-added at each change of media 1X or 2X final concentration , depending on the cell type and considering the size and growth rate. Thus, the substrate has not been considered a limiting factor even at lower concentration Supplementary Fig. After incubation, RLU were remeasured using the luminometer. The study was approved by the Ethical and Institutional Review Board of the University Hospital of Modena and was performed in accordance with the relevant guidelines and regulations.
Tumor specimens were obtained after obtaining signed informed consent from patients who underwent surgery at the Chest Surgery Division University Hospital of Modena.
Immunophenotyping of cells recovered after tumor tissue digestion was performed. All animal experiments were approved by the Animal Ethics Committee of Modena and all methods were performed in accordance with the relevant guidelines and regulations. Tumor implantation and cell delivery were performed as described in the Results section. Antoni, D.
In vitro sensitivity assays in cancer: A review, analysis, and prognosis
Fitzgerald, K. A two-sample t-test for unpaired data was performed to compare data sets. A total of 18 anti-proliferative agents were selected based on their anti-proliferative mechanistic diversity and were tested in the Triticum aestivum root elongation assay. The values obtained for the inhibitory effects are presented in Table I. The values ranged from The inhibitory effect on wheat root elongation after 24 h of exposure to the anti-proliferative agents. As shown in Table II , the Kruskal Wallis test revealed statistically significant differences between the results obtained at various concentrations, although a linear effect-concentration association was not found for all determinations.
No association was found between concentration and root elongation for albendazole, chlorambucil, hydroxyurea, imatinib and irinotecan. Dunn's post-test revealed statistically significant differences between the results obtained for each concentration and the solvent control sample, with the exception of quinine and verapamil, which induced a stimulating effect, without being statistically significant. NT, not tested.
serbiansingingfederation.org/images/soire/4534-site-de.php Detecting only 5 out of the 18 anti-proliferative compounds tested suggests a poor predictive power and a high false-negative ratio. For all these compounds, the half maximal inhibitory concentration IC50 values and their corresponding negative logarithm pIC50 were computed based on the dose-response curves presented in Fig. The IC50 values, their corresponding confidence interval, and correlation coefficient r 2 were calculated and are presented in Table III in ascending order.
Inhibition curves of wheat root elongation following treatment for 24 h with: A methotrexate, B cantharidin, C colchicine, D indoleacetic acid, E cisplatin, F aminophylline, and G verapamil. The high rate of false-positives is due to the small tested set and the deliberate selection of indoleacetic acid as a known root growth inhibitor without an anti-proliferative effect.
This is probably the main reason for the low sensitivity of the test. In order to better determine the wheat root inhibition profile of all the 20 tested substances, a hierarchical cluster analysis was performed using the furthest neighbor method and Euclidean distance measure. Three major clusters were obtained Fig.