scholarly journals Swift Formation of Optimal Single Spheroids towards In-Vitro 3-Dimensional Tumour Models

2021 ◽  
Author(s):  
Stephanie Eugenie Brigitte McArdle ◽  
Kinana Habra ◽  
Joshua R D Pearson
Keyword(s):  
Cancer Cells ◽  
3D Structure ◽  
Proliferating Cells ◽  
Monolayer Cell Culture ◽  
3 Dimensional ◽  
Rapid Generation ◽  
The Cost ◽  
High Throughput Experiments

Monolayer cell culture, while useful for basic in vitro studies, are not physiologically relevant. Spheroids, on the other hand provide a more complex 3-dimensional (3D) structure which more resemble the in vivo tumour growth thereby allowing results obtained with those on proliferation, cell death, differentiation, metabolism, and various anti-tumour therapies to be more predictive of in vivo outcomes. However, the cost associated with their generation often involve expensive, plate, media, and growth supplements, which have limited their use for high throughput experiments. The protocol herein presents a novel and rapid generation for single spheroids of various cancer cell lines, U87 MG; SEBTA-027; SF188, brain cancer cells, DU-145, TRAMP-C1, prostate cancer cells, in 96-round bottom well plates. Cells are washed with anti-adherent solution, and the homogeneous compact spheroid morphology was evidenced as early as 24 hours after 10 minutes centrifugation for the seeded cells. By using confocal microscopy, the proliferating cells were traced in the rim and the dead cells were found inside the core region of the spheroid. The H&E stain of spheroid slices and the western blotting were utilised to investigate the tightness of the cell packaging by adhesion proteins. Carnosine was used as an example of treatment for U87 single spheroids. The protocol allows the rapid generation of spheroids, which will help towards reducing the number of tests performed on animals.

scholarly journals FSMP-09. FORMATE PROMOTES CANCER CELL INVASION AND METASTASIS VIA CALCIUM SIGNALING

2020 ◽  
Vol 3 (Supplement_1) ◽  
pp. i18-i18
Author(s):  
Catherine Delbrouck ◽  
Vitaly I Pozdeev ◽  
Anais Oudin ◽  
Kamil Grzyb ◽  
Laura Neises ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Ex Vivo ◽  
Tumor Model ◽  
Underlying Mechanism ◽  
Tumor Escape ◽  
Proliferating Cells ◽  
Nucleotide Synthesis ◽  
Formate Production

Abstract Serine catabolism via the folate cycle provides formate that is essential for nucleotide synthesis in proliferating cells. In addition to this canonical function to support biomass production in anabolic cells, we have recently demonstrated in vitro and in vivo that formate production in cancer cells is often in excess of the anabolic demand. This excess formate production is characterized by formate overflow and thus, net formate excretion into the tumor microenvironment. Interestingly, we observe increased rates of formate overflow upon different chemical perturbations that induce growth arrest. Thus, stressed cancer cells that encounter growth restriction such as upon chemotherapy, are often characterized by increased formate release rates. We demonstrated that such high formate levels in the extracellular space promote invasion of glioblastoma cells. Using ex vivo brain slice cultures and an orthotopic brain tumor model, we demonstrate that silencing MTHFD1L, the essential enzyme to enable formate overflow, results in decreased invasiveness of the tumor. Embarking from this observation, we investigated the underlying mechanism and now provide evidence that the formate-dependent increase of cell motility is mediated by an activation of Ca2+ signaling. Activation of Ca2+ signaling triggers integrin and matrix metallopeptidase (MMP) responses enabling the invasion process. Targeting either the Ca2+ response or MMP release can suppress the formate dependent increase in invasion. Finally, we tested the effect of formate also in context of breast cancer where we were able to recapitulate our observation of increased invasiveness and, in this case, formate also promoted the metastatic potential. We conclude that excreted formate might serve as a cellular stress signal that represents a promotive trigger to support tumor escape mechanisms.

scholarly journals Magnetic Compression of Tumor Spheroids Increases Cell Proliferation In Vitro and Cancer Progression In Vivo

Cancers ◽  
2022 ◽  
Vol 14 (2) ◽  
pp. 366
Author(s):  
Gaëtan Mary ◽  
Brice Malgras ◽  
Jose Efrain Perez ◽  
Irène Nagle ◽  
Nathalie Luciani ◽  
...  
Keyword(s):  
Colon Cancer ◽  
Cancer Cells ◽  
Cancer Progression ◽  
Peritoneal Cancer Index ◽  
Colon Cancer Cells ◽  
Tumor Spheroids ◽  
Proliferating Cells ◽  
The Impact

A growing tumor is submitted to ever-evolving mechanical stress. Endoscopic procedures add additional constraints. However, the impact of mechanical forces on cancer progression is still debated. Herein, a set of magnetic methods is proposed to form tumor spheroids and to subject them to remote deformation, mimicking stent-imposed compression. Upon application of a permanent magnet, the magnetic tumor spheroids (formed from colon cancer cells or from glioblastoma cells) are compressed by 50% of their initial diameters. Such significant deformation triggers an increase in the spheroid proliferation for both cell lines, correlated with an increase in the number of proliferating cells toward its center and associated with an overexpression of the matrix metalloproteinase−9 (MMP−9). In vivo peritoneal injection of the spheroids made from colon cancer cells confirmed the increased aggressiveness of the compressed spheroids, with almost a doubling of the peritoneal cancer index (PCI), as compared with non-stimulated spheroids. Moreover, liver metastasis of labeled cells was observed only in animals grafted with stimulated spheroids. Altogether, these results demonstrate that a large compression of tumor spheroids enhances cancer proliferation and metastatic process and could have implications in clinical procedures where tumor compression plays a role.

Preclinical activity of BC2059, a novel compound that degrades beta-catenin, in colon cancer models.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. e14090-e14090
Author(s):  
Sunil Sharma ◽  
Raffaella Soldi ◽  
Glynn Weldon Gilcrease ◽  
Stephen Horrigan
Keyword(s):  
Colon Cancer ◽  
Cancer Cells ◽  
Tumor Invasion ◽  
Tumor Type ◽  
Dependent Manner ◽  
3 Dimensional ◽  
Cancer Models ◽  
Ht 29

e14090 Background: BC2059 (BetaCat Pharmaceuticals) is a novel small molecule that degrades ß-catenin in a proteosomally dependent manner in cancer cells. Methods: We tested the activity of BC2059 in vitro, in patient derived colon cancers in 3-dimensional cultures and in vivo using HT-29 xenografts. We also used a novel doxycycline driven siRNA model that downregulates ß-catenin to test activity of BC2059. Results: BC2059 was highly active with IC90s in MTS assays (μM): SW480 (0.44), Colo205 (0.66), Colo201 (0.03), HT29 (0.92) and relatively inactive in SW48 (9.83) or L cells (normal) (4). In a 3-Dimensional culture system where we grew patient-derived colon cancer cells in a matrigel system and measured cell death by ethidium bromide exclusion, surprisingly, the average EC50 was 200 picomolar indicating that the compound was much more effective in patient derived samples. In matrigel tumor invasion assays, BC2059 demonstrated potent inhibition of tumor invasion. In HT-29 mouse xenografts, BC2059 series demonstrated tumor growth inhibition with no weight loss with depletion of ß-catenin. We also used chromatin immunoprecipitation (CHIP) assays to demonstrate depletion of ß-catenin on myc and axin promoters. In combination experiments, BC2059 displayed synergy in vitro with 5-fluorouracil in vitro. We used a doxycycline driven siRNA system in colon cancer cell lines and show that the compound activity is synergistic with depletion of ß-catenin. We also conducted combination of BC2059 series of compounds using a proteasome inhibitor MG132 and demonstrate reversal of activity with proteasome inhibition. Conclusions: In conclusion, BC2059 demonstrates potent activity in vitro and in vivo against colon cancer models singly or in combination. Further, it is surprisingly potent in patient derived colon cancer models. It appears to degrade ß-catenin in a proteosomally dependent manner on important effectors like myc and axin. It merits further evaluation in this tumor type.

scholarly journals Discovery and optimization of piperazine-1-thiourea-based human phosphoglycerate dehydrogenase inhibitors

2018 ◽  
Author(s):  
Jason M. Rohde ◽  
Kyle R. Brimacombe ◽  
Li Liu ◽  
Michael E. Pacold ◽  
Adam Yasgar ◽  
...  
Keyword(s):  
Cancer Cells ◽  
High Throughput Screening ◽  
Feedback Inhibition ◽  
Proliferating Cells ◽  
Important Amino Acid ◽  
Rate Limiting Step ◽  
Phosphoglycerate Dehydrogenase ◽  
Hit Validation

AbstractProliferating cells, including cancer cells, obtain serine both exogenously and via the metabolism of glucose. By catalyzing the first, rate-limiting step in the synthesis of serine from glucose, phosphoglycerate dehydrogenase (PHGDH) controls flux through the biosynthetic pathway for this important amino acid and represents a putative target in oncology. To discover inhibitors of PHGDH, a coupled biochemical assay was developed and optimized to enable high-throughput screening for inhibitors of human PHGDH. Feedback inhibition was minimized by coupling PHGDH activity to two downstream enzymes (PSAT1 and PSPH), providing a significant improvement in enzymatic turnover. Further coupling of NADH to a diaphorase/resazurin system enabled a red-shifted detection readout, minimizing interference due to compound autofluorescence. With this protocol, over 400,000 small molecules were screened for PHGDH inhibition, and following hit validation and triage work, a piperazine-1-thiourea was identified. Following rounds of medicinal chemistry and SAR exploration, two probes (NCT-502 and NCT-503) were identified. These molecules demonstrated improved target activity and encouraging ADME properties, enabling both in vitro and in vivo assessment of the biological importance of PHGDH, and its role in the fate of serine in PHGDH-dependent cancer cells.

scholarly journals In Vitro and In Vivo Characterization of MCT1 Inhibitor AZD3965 Confirms Preclinical Safety Compatible with Breast Cancer Treatment

Cancers ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 569
Author(s):  
Zohra Benyahia ◽  
Marine C. N. M. Blackman ◽  
Loïc Hamelin ◽  
Luca X. Zampieri ◽  
Tania Capeloa ◽  
...  
Keyword(s):  
Cancer Cells ◽  
New Drugs ◽  
Phenotypic Characterization ◽  
Memory Retention ◽  
Proliferating Cells ◽  
Brain Physiology ◽  
Metabolic Heterogeneity

To survive and proliferate in solid tumors, cancer cells adapt and evolve rapidly in microenvironments where oxygen and substrate bioavailability fluctuates over time and space. This creates metabolic heterogeneity. Cancer cells can further cooperate metabolically, for example by swapping glycolytic end-product lactate for blood-borne glucose. This type of cooperation can be targeted therapeutically, since transmembrane lactate exchanges are facilitated by lactate-proton symporters of the monocarboxylate (MCT) family. Among new drugs, AZD3965 is a first-in-class selective MCT1 inhibitor currently tested in Phase I/II clinical trials for patients with different types of cancers. Because MCT1 can function bidirectionally, we tested here whether and how malignant and nonmalignant cells adapt their metabolism and MCT repertoire when AZD3965 inhibits either lactate import or export. Using breast-associated malignant and nonmalignant cell lines as models, we report that AZD3965 is not directly cytotoxic. In the presence of glucose and glutamine, oxidative cells can survive when lactate uptake is blocked, and proliferating cells compensate MCT1 inhibition by overexpressing MCT4, a specialized facilitator of lactate export. Phenotypic characterization of mice focusing on metabolism, muscle and brain physiology found partial and transient memory retention defect as sole consequence of MCT1 inhibition by AZD3965. We therefore conclude that AZD3965 is compatible with anticancer therapy.

ECONOMIC PERSPECTIVES OF BIOTECHNOLOGIES USING IN ANIMAL FARMING

1970 ◽  
pp. 57-60
Author(s):  
I. F. Gorlov ◽  
A. A. Mosolov ◽  
G. V. Komlatskiy ◽  
M. A. Nesterenko ◽  
K. D. Nimbona ◽  
...  
Keyword(s):  
Embryo Transfer ◽  
Economic Effect ◽  
Dairy Herd ◽  
State Level ◽  
Modern Level ◽  
Economic Perspectives ◽  
The Cost ◽  
Short Time

The article presents materials on the study of the possibility of reproduction and increase in the herd of highly productive cows through the use of embryo transplantation technology. The classical (in vivo) and more modern, developing (in vitro) methods of embryotransfer, their positive and negative sides are considered in detail. The possibility of accelerating the breeding process by using the method of transplantation, in which from one cow can be obtained from 10 to 100 calves, which will allow for 4-5 years, almost any herd (of any size and breed) with the help of biotechnology to turn into a cattle-breeding enterprise of the most modern level. At the same time, heifers obtained from unproductive cows can be used as "surrogate" mothers who are transplanted with the best donor embryos, which allows to obtain a full-fledged offspring adapted to local environmental conditions. A detailed scheme of obtaining, evaluation, storage, as well as the cost and economic effect of embryo transplantation was calculated, the market was evaluated, the required annual volume of transplants and the number of donor cows for large livestock farms were determined. As a positive example of "Scientific-production enterprise "Centre of biotechnology and embryo transfer" in 2014, implemented a project for accelerated replacement and genetic improvement of the dairy herd, engraftment averaged 57-69%, and the economic effect of the enterprise from getting a single animal by the method of embryo transfer, compared with imports of similar close in quality, ranged from 60 to 100 thousand rubles on his head. It is shown that it is necessary to organize at the state level a developed service for embryo transplantation to reduce the cost of embryo transfer and the possibility of creating in a short time in the country's own highly productive breeding nucleus of dairy and beef cattle, which will reduce, and in the future completely eliminate, import dependence on cattle products.

In Vitro and In Vivo Antitumor Efficacy of Docetaxel and Sorafenib Combination in Human Pancreatic Cancer Cells

2010 ◽  
Vol 999 (999) ◽  
pp. 1-11
Author(s):  
P. Ulivi ◽  
C. Arienti ◽  
W. Zoli ◽  
M. Scarsella ◽  
S. Carloni ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Cancer Cells ◽  
Antitumor Efficacy ◽  
Human Pancreatic Cancer ◽  
Pancreatic Cancer Cells

A Novel Triazole Derivative Drug Presenting In Vitro and In Vivo Anticancer Properties

2018 ◽  
Vol 18 (17) ◽  
pp. 1483-1493
Author(s):  
Ricardo Imbroisi Filho ◽  
Daniel T.G. Gonzaga ◽  
Thainá M. Demaria ◽  
João G.B. Leandro ◽  
Dora C.S. Costa ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Cell Line ◽  
Cancer Cell ◽  
Cancer Cell Line ◽  
Hepatic Function ◽  
Anticancer Properties ◽  
Mcf 7

Background: Cancer is a major cause of death worldwide, despite many different drugs available to treat the disease. This high mortality rate is largely due to the complexity of the disease, which results from several genetic and epigenetic changes. Therefore, researchers are constantly searching for novel drugs that can target different and multiple aspects of cancer. Experimental: After a screening, we selected one novel molecule, out of ninety-four triazole derivatives, that strongly affects the viability and proliferation of the human breast cancer cell line MCF-7, with minimal effects on non-cancer cells. The drug, named DAN94, induced a dose-dependent decrease in MCF-7 cells viability, with an IC50 of 3.2 ± 0.2 µM. Additionally, DAN94 interfered with mitochondria metabolism promoting reactive oxygen species production, triggering apoptosis and arresting the cancer cells on G1/G0 phase of cell cycle, inhibiting cell proliferation. These effects are not observed when the drug was tested in the non-cancer cell line MCF10A. Using a mouse model with xenograft tumor implants, the drug preventing tumor growth presented no toxicity for the animal and without altering biochemical markers of hepatic function. Results and Conclusion: The novel drug DAN94 is selective for cancer cells, targeting the mitochondrial metabolism, which culminates in the cancer cell death. In the end, DAN94 has been shown to be a promising drug for controlling breast cancer with minimal undesirable effects.

scholarly journals A New Smoothened Antagonist Bearing the Purine Scaffold Shows Antitumour Activity In Vitro and In Vivo

2021 ◽  
Vol 22 (16) ◽  
pp. 8372
Author(s):  
Ana María Zárate ◽  
Christian Espinosa-Bustos ◽  
Simón Guerrero ◽  
Angélica Fierro ◽  
Felipe Oyarzún-Ampuero ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Human Cancer ◽  
Antitumour Activity ◽  
Anticancer Compounds ◽  
Human Cancer Cells ◽  
Cycle Arrest ◽  
Apoptosis Inducer ◽  
Purine Ring

The Smoothened (SMO) receptor is the most druggable target in the Hedgehog (HH) pathway for anticancer compounds. However, SMO antagonists such as vismodegib rapidly develop drug resistance. In this study, new SMO antagonists having the versatile purine ring as a scaffold were designed, synthesised, and biologically tested to provide an insight to their mechanism of action. Compound 4s was the most active and the best inhibitor of cell growth and selectively cytotoxic to cancer cells. 4s induced cell cycle arrest, apoptosis, a reduction in colony formation and downregulation of PTCH and GLI1 expression. BODIPY-cyclopamine displacement assays confirmed 4s is a SMO antagonist. In vivo, 4s strongly inhibited tumour relapse and metastasis of melanoma cells in mice. In vitro, 4s was more efficient than vismodegib to induce apoptosis in human cancer cells and that might be attributed to its dual ability to function as a SMO antagonist and apoptosis inducer.

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