Direct visualization of emergent metastatic features within an ex vivo model of the tumor microenvironment.

Preventing tumor cells from acquiring metastatic properties would significantly reduce cancer mortality. However, due to the complex nature of this process, it remains one of the most poorly understood and untreatable aspects of cancer. Ischemia and hypoxia in solid tumors are requisite in metastasis formation -- conditions that arise far from functional blood vessels and deep within tumor tissues. These secluded locations impede the observation of pre-metastatic tumor cells and their interactions with stromal cells, which are also critical in the initiation of this process. Thus, the initiation of metastasis has been incredibly difficult to model in the lab and to observe in vivo. We present an ex vivo model of the tumor microenvironment, called 3MIC, which overcomes these experimental challenges and enables the observation of ischemic tumor cells in their native 3D context with high spatial and temporal resolutions. The 3MIC recreates ischemic conditions in the tumor microenvironment and facilitates the co-culture of different cell types. Using live microscopy, we showed that ischemia, but not hypoxia alone, increases the motility and invasive properties of cells derived from primary tumors. These changes are phenotypic and can occur without clonal selection. We directly observed how interactions with stromal cells such as macrophages increased tumor invasion in conjunction with the effects of an ischemic microenvironment. Finally, we tested the effects of chemotherapy drugs under different metabolic microenvironments and found that ischemic tumor cells are more resistant to paclitaxel, possibly due to a metabolic resistance mechanism. Overall, the 3MIC is a cost-effective system that allows for the dissection of the complexity of the tumor microenvironment and direct observation of the emergence of metastasis, as well as the testing of treatments that may halt this process.

Phospholipid Mediator Induced Transformation in Three-Dimensional Cultures.

Several models have been developed to study cancer, such as rodent models and established cell lines. Valuable insights into carcinogenesis have been provided by studies using these models. Cell lines have provided an understanding of the deregulation of molecular signaling associated with breast tumorigenesis, while rodent models are widely used to study cellular and molecular characteristics of breast cancer in vivo. The establishment of 3D cultures of breast epithelial and cancerous cells aids in bridging the gap between in vivo and in vitro models by mimicking the in vivo conditions in vitro. This model can be used to understand the deregulation of complex molecular signaling events and the cellular characteristics during breast carcinogenesis. Here, a 3D culture system is modified to study a phospholipid mediator-induced (Platelet Activating Factor, PAF) transformation. Immunomodulators and other secreted molecules play a major role in tumor initiation and progression in the breast. In the present study, 3D acinar cultures of breast epithelial cells are exposed to PAF exhibited transformation characteristics such as loss of polarity and altered cellular characteristics. This 3D culture system will assist in shedding light on genetic and/or epigenetic perturbations induced by various small molecule entities in the tumor microenvironment. Additionally, this system will also provide a platform for the identification of novel as well as known genes that may be involved in the process of transformation.

The MEMIC is an ex vivo system to model the complexity of the tumor microenvironment.

There is an urgent need for accurate, scalable and cost-efficient models of the tumor microenvironment. Here, we detail how to fabricate and use the metabolic microenvironment chamber (MEMIC) - a 3D-printed ex vivo model of intratumoral heterogeneity. A major driver of the cellular and molecular diversity in tumors is accessibility to the blood stream. Whereas perivascular tumor cells have direct access to oxygen and nutrients, cells further from the vasculature must survive under progressively more ischemic environments. The MEMIC simulates this differential access to nutrients, allow co-culturing any number of cell types, and it is optimized for live imaging and other microscopy-based analyses. Owing to a modular design and full experimental control, the MEMIC provides insights into the tumor microenvironment that would be difficult to obtain via other methods. As proof of principle, we show that cells sense gradual changes in metabolite concentration leading to predictable molecular and cellular spatial patterns. We propose the MEMIC as a complement to standard in vitro and in vivo experiments, diversifying the tools available to accurately model, perturb and monitor the tumor microenvironment.

Prolonged Exposure to Platelet Activating Factor Transforms Breast Epithelial Cells.

Lipid species are known to have various biological functions owing to their structural differences, and each of them possesses a specific role to play depending upon their location and distribution in the cell. Some of these lipids interact with proteins on the cell membrane and acts as second messengers. The level of lipid mediators is generally maintained in the cell by feedback mechanisms; however, their improper degradation or enhanced production leads to their accumulation in the tumor microenvironment and disturbs the homeostasis of the cell. Platelet activating factor (PAF) is a known phospholipid mediator secreted upon immunological challenges by platelets, neutrophils, basophils, and macrophages. PAF, as a potent inflammatory molecule, is well studied, and its role in various cancers and cardiovascular diseases has also been investigated. Interestingly, increased levels of PAF have been found in the blood plasma of smokers, and breast cancer cells have shown the accumulation of PAF in presence of cigarette smoke extract. This accumulation was found to increase tumor cell motility that in turn could promote metastasis. Beyond this, however, the effect of PAF on tumorigenesis has not yet been well explored. Here, we show that the continuous exposure of 3D breast acinar cultures to PAF resulted in the activation of various oncogenic signaling pathways leading to transformation. We also found that the presence of PAF in the micro-environment increased the expression of PAF receptor (PAF-R), which corroborated with the higher expression of PAF-R detected in some epithelial cancers, as per literature. Thus, this study impresses on the fact that the presence of PAF alters the cellular microenvironment and eventually triggers irreversible effects that can cumulatively lead to transformation.

DNA-dependent protein kinase plays a central role in transformation of breast epithelial cells following alkylation damage.

DNA alkylating agents form the first line of cancer chemotherapy. They not only kill cells but also behave as potential carcinogens. MNU, a DNA methylating agent, is well known to induce mammary tumours in rodents. However, the mechanism of tumorigenesis is not well understood. Our study reports a novel role played by DNA-dependent protein kinase (DNA-PK) in methylation damage-induced transformation using three-dimensional breast acinar cultures. Here, we report that exposure of breast epithelial cells to MNU inhibited polarisation at the basolateral domain, increased dispersal of the Golgi at the apical domain and induced an epithelial-to-mesenchymal transition (EMT)-like phenotype as well as invasion. This altered Golgi phenotype correlated with impaired intracellular trafficking. Inhibition of DNA-PK resulted in almost complete reversal of the altered Golgi phenotype and partial rescue of the polarity defect and EMT-like phenotype. The results confirm that methylation damage-induced activation of DNA-PK is a major mechanism in mediating cellular transformation.This article has an associated First Person interview with the first author of the paper.

Drug-Triggered Self-Assembly of Linear Polymer into Nanoparticles for Simultaneous Delivery of Hydrophobic and Hydrophilic Drugs in Breast Cancer Cells.

Breast cancer is the most devastating disease among females globally. Conventional chemotherapeutic regimen relies on the use of highly cytotoxic drugs as monotherapy and combination therapy leading to severe side effects to the patients as collateral damage. Moreover, combining hydrophobic and hydrophilic drugs create erratic biodistribution and suboptimal medicinal outcome. Hence, packaging multiple drugs of diverse mechanisms of action and biodistribution for safe delivery into tumor tissues with optimal dosages is indispensable for next-generation breast cancer therapy. To address these, in this report, we describe a unique cisplatin-triggered self-assembly of linear polymer into 3D-spherical sub 200 nm particles. These nanoparticles comprise a hydrophobic (paclitaxel) and hydrophilic drug (cisplatin) simultaneously in a single particle. Molecular dynamics simulation revealed hydrophilic-hydrophilic interaction and interchain H-bonding as underlying mechanisms of self-assembly. Confocal microscopy studies evidently demonstrated that these novel nanoparticles can home into lysosomes in breast cancer cells, fragment subcellular nuclei, and prevent cell division, leading to improved breast cancer cell death compared to free drug combination. Moreover, 3D-breast tumor spheroids were reduced remarkably by the treatment of these nanoparticles within 24 h. These dual-drug-loaded self-assembled polymeric nanoparticles have prospective to be translated into a clinical strategy for breast cancer patients.