Development of a 3D in vitro model of cancer progression

The hallmark of cancer is the ability of cancer cells to disseminate and invade to distant organs. Metastasis accounts for over 90% of cancer related deaths and therefore requires a better understanding of the metastatic cascade to develop better therapeutic regimens. The metastatic process is heavily influenced by the extracellular matrix (ECM) density and composition of the surrounding tumour microenvironment. These microenvironmental cues also regulate the angiogenic processes within a tumour, facilitating the spread of cancer cells. The aim of this thesis was to develop a biomimetic 3D in vitro model of colorectal cancer with controllable matrix parameters that regulate cancer invasion, the formation of a primitive vascular network and response to targeted drug treatment. A novel 3D in vitro cancer model was established based on the removal of interstitial fluid in collagen type I hydrogels. Colorectal cancer cells cultured in dense collagen gels formed well-defined cellular aggregates mimicking avascular micrometastases observed in vivo. Cancer cells invaded from the artificial cancer mass (ACM) into the stromal surround in cell specific patterns, either as spherical aggregates or cell sheets. Invasion into a denser collagen matrix altered the migration pattern predominantly to cell sheets. Laminin was found to enhance the invasion profile of colorectal cancer cells independent of stromal matrix density. The pro-invasive and epithelial-mesenchymal transition (EMT) markers MMP7 and vimentin were upregulated in 3D cultures in comparison to 2D monolayers. Stromal cultures containing fibroblasts and endothelial cells formed highly branched end-to-end vascular networks in the presence of laminin. The addition of cancer cells produces significantly longer but substantially less interconnected vascular networks mimicking in vivo ‘leaky’ tumour vasculature. The loss of CK20 by invading cancer cells significantly correlated with the overall distance of invasion into the stromal surround. Although the expression of the biomarker EGFR was upregulated in 3D, targeted treatment using cetuximab lead to a greater inhibition profile in 2D monolayers than in 3D cultures. Drug resistance in 3D cultures corresponded with the presence of cancer stem cells. These findings signify the importance of 3D in vitro cancer models as important tools to study the effect of microenvironmental conditions on tumour malignancy

Efficacy of DOPE/DC-cholesterol liposomes and GCPQ micelles as AZD6244 nanocarriers in a 3D colorectal cancer in vitro model

Aim: In this work, we use cationic organic nanocarriers as chemotherapy delivery platforms and test them in a colorectal cancer 3D in vitro model. Materials & methods: We used 3beta-(N-[N′,N′-dimethylaminoethane]carbamoyl])cholesterol (DC-chol) and dioleoylphosphatidylethanolamine (DOPE) liposomes and N-palmitoyl-N-monomethyl-N,N-dimethyl-N,N,N-trimethyl-6-O-glycolchitosan (GCPQ) micelles, to deliver AZD6244, a MEK inhibitor, to HCT116 cells cultured as monolayers and in 3D in vitro cancer models (tumoroids). Results: Nanoparticle-mediated drug delivery was superior to the free drug in monolayer experiments and despite their therapeutic effect being hindered by poor diffusion through the cancer mass, GCPQ micelles were also superior in tumoroids. Conclusion: These results support the role of nanoparticles in improving drug delivery and highlight the need to include 3D cancer models in early phases of drug development

Efficacy of DOPE/DC-cholesterol liposomes and GCPQ micelles as AZD6244 nanocarriers in a 3D colorectal cancer in vitro model

AIM: In this work, we use cationic organic nanocarriers as chemotherapy delivery platforms and test them in a colorectal cancer 3D in vitro model. MATERIALS & METHODS: We used 3beta-(N-[N',N'-dimethylaminoethane]carbamoyl])cholesterol (DC-chol) and dioleoylphosphatidylethanolamine (DOPE) liposomes and N-palmitoyl-N-monomethyl-N,N-dimethyl-N,N,N-trimethyl-6-O-glycolchitosan (GCPQ) micelles, to deliver AZD6244, a MEK inhibitor, to HCT116 cells cultured as monolayers and in 3D in vitro cancer models (tumoroids). RESULTS: Nanoparticle-mediated drug delivery was superior to the free drug in monolayer experiments and despite their therapeutic effect being hindered by poor diffusion through the cancer mass, GCPQ micelles were also superior in tumoroids. CONCLUSION: These results support the role of nanoparticles in improving drug delivery and highlight the need to include 3D cancer models in early phases of drug development

Engineering a vascularised 3D in vitro model of cancer progression.

The hallmark of tumours is the ability of cancerous cells to promote vascular growth, to disseminate and invade to distant organs. The metastatic process is heavily influenced by the extracellular matrix (ECM) density and composition of the surrounding tumour microenvironment. These microenvironmental cues, which include hypoxia, also regulate the angiogenic processes within a tumour, facilitating the spread of cancer cells. We engineered compartmentalized biomimetic colorectal tumouroids with stromal surrounds that comprised a range of ECM densities, composition and stromal cell populations. Recapitulating tissue ECM composition and stromal cell composition enhanced cancer cell invasion. Manipulation of ECM density was associated with an altered migration pattern from glandular buds (cellular aggregates) to epithelial cell sheets. Laminin appeared to be a critical component in regulating endothelial cell morphology and vascular network formation. Interestingly, the disruption of vascular networks by cancer cells was driven by changes in expression of several anti-angiogenic genes. Cancer cells cultured in our biomimetic tumouroids exhibited intratumoural heterogeneity that was associated with increased tumour invasion into the stroma. These findings demonstrate that our 3D in vitro tumour model exhibits biomimetic attributes that may permit their use in studying microenvironment clues of tumour progression and angiogenesis

Cancer invasion regulates vascular complexity in a three-dimensional biomimetic model

Introduction There is a growing appreciation for including a complex, vascularised stroma in three-dimensional (3D) tumour models to recapitulate the native tumour microenvironment in situ. Methods Using a compartmentalised, biomimetic, 3D cancer model, comprising a central cancer mass surrounded by a vascularised stroma, we have tested the invasive capability of colorectal cancer cells. Results We show histological analysis of dense collagen I/laminin scaffolds, forming necrotic cores with cellular debris. Furthermore, cancer cells within this 3D matrix form spheroids, which is corroborated with high EpCAM expression. We validate the invasive growth of cancer cells into the stroma through quantitative image analysis and upregulation of known invasive gene markers, including metastasis associated in colon cancer 1, matrix metalloproteinase 7 and heparinase. Tumouroids containing highly invasive HCT116 cancer masses form less complex and less branched vascular networks, recapitulating ‘leaky’ vasculature associated with highly metastatic cancers. Angiogenic factors regulating this were vascular endothelial growth factor A and hepatocyte growth factor active protein. Where vascular networks were formed with less invasive cancer masses (HT29), higher expression of vascular endothelial cadherin active protein resulted in more complex and branched networks. To eliminate the cell–cell interaction between the cancer mass and stroma, we developed a three-compartment model containing an acellular ring to test the chemoattractant pull from the cancer mass. This resulted in migration of endothelial networks through the acellular ring accompanied by alignment of vascular networks at the cancer/stroma boundary. Discussion This work interrogates to the gene and protein level how cancer cells influence the development of a complex stroma, which shows to be directly influenced by the invasive capability of the cancer

The efficacy of cetuximab in a tissue-engineered three-dimensional in vitro model of colorectal cancer.

The preclinical development process of chemotherapeutic drugs is often carried out in two-dimensional monolayer cultures. However, a considerable amount of evidence demonstrates that two-dimensional cell culture does not accurately reflect the three-dimensional in vivo tumour microenvironment, specifically with regard to gene expression profiles, oxygen and nutrient gradients and pharmacokinetics. With this objective in mind, we have developed and established a physiologically relevant three-dimensional in vitro model of colorectal cancer based on the removal of interstitial fluid from collagen type I hydrogels. We employed the RAFT™ (Real Architecture For 3D Tissue) system for producing three-dimensional cultures to create a controlled reproducible, multiwell testing platform. Using the HT29 and HCT116 cell lines to model epidermal growth factor receptor expressing colorectal cancers, we characterized three-dimensional cell growth and morphology in addition to the anti-proliferative effects of the anti-epidermal growth factor receptor chemotherapeutic agent cetuximab in comparison to two-dimensional monolayer cultures. Cells proliferated well for 14 days in three-dimensional culture and formed well-defined cellular aggregates within the concentrated collagen matrix. Epidermal growth factor receptor expression levels revealed a twofold and threefold increase in three-dimensional cultures for both HT29 and HCT116 cells in comparison to two-dimensional monolayers, respectively (p < 0.05; p < 0.01). Cetuximab efficacy was significantly lower in HT29 three-dimensional cultures in comparison to two-dimensional monolayers, whereas HCT116 cells in both two-dimension and three-dimension were non-responsive to treatment in agreement with their KRAS mutant status. In summary, these results confirm the use of a three-dimensional in vitro cancer model as a suitable drug-screening platform for in vitro pharmacological testing

Dihydrophenazine:a multifunctional new weapon that kills multidrug-resistant Acinetobacter baumannii and restores carbapenem and oxidative stress susceptibilities

AimsThe current work aims to fully characterize a new antimicrobial agent against Acinetobacter baumannii, which continues to represent a growing threat to healthcare settings worldwide. With minimal treatment options due to the extensive spread of resistance to almost all the available antimicrobials, the hunt for new antimicrobial agents is a high priority. Methods and resultsAn Egyptian soil-derived bacterium strain NHM-077B proved to be a promising source for a new antimicrobial agent. Bioguided fractionation of the culture supernatants of NHM-077B followed by chemical structure elucidation identified the active antimicrobial agent as 1-hydroxy phenazine. Chemical synthesis yielded more derivatives, including dihydrophenazine (DHP), which proved to be the most potent against A. baumannii, yet it exhibited a safe cytotoxicity profile against human skin fibroblasts. Proteomics analysis of the cells treated with DHP revealed multiple proteins with altered expression that could be correlated to the observed phenotypes and potential mechanism of the antimicrobial action of DHP. DHP is a multi-pronged agent that affects membrane integrity, increases susceptibility to oxidative stress, interferes with amino acids/protein synthesis, and modulates virulence-related proteins. Interestingly, DHP in sub-inhibitory concentrations resensitizes the highly virulent carbapenem-resistant A. baumannii strain AB5075 to carbapenems providing great hope in regaining some of the benefits of this important class of antibiotics. ConclusionsThis work underscores the potential of DHP as a promising new agent with multifunctional roles as both a classical and non-conventional antimicrobial agent that is urgently needed.<br/