A 3D adrenocortical carcinoma tumor platform for preclinical modeling of drug response and matrix metalloproteinase activity.

Adrenocortical carcinoma (ACC) has a poor prognosis, and no new drugs have been identified in decades. The absence of drug development can partly be attributed to a lack of preclinical models. Both animal models and 2D cell cultures of ACC fail to accurately mimic the disease, as animal physiology is inherently different than humans, and 2D cultures fail to represent the crucial 3D architecture. Organoids and other small 3D in vitro models of tissues or tumors can model certain complexities of human in vivo biology; however, this technology has largely yet to be applied to ACC. In this study, we describe the generation of 3D tumor constructs from an established ACC cell line, NCI-H295R. NCI-H295R cells were encapsulated to generate 3D ACC constructs. Tumor constructs were assessed for biomarker expression, viability, proliferation, and cortisol production. In addition, matrix metalloproteinase (MMP) functionality was assessed directly using fluorogenic MMP-sensitive biosensors and through infusion of NCI-H295R cells into a metastasis-on-a-chip microfluidic device platform. ACC tumor constructs showed expression of biomarkers associated with ACC, including SF-1, Melan A, and inhibin α. Treatment of ACC tumor constructs with chemotherapeutics demonstrated decreased drug sensitivity compared to 2D cell culture. Since most tumor cells migrate through tissue using MMPs to break down extracellular matrix, we validated the utility of ACC tumor constructs by integrating fluorogenic MMP-sensitive peptide biosensors within the tumor constructs. Lastly, in our metastasis-on-a-chip device, NCI-H295R cells successfully engrafted in a downstream lung cell line-based construct, but invasion distance into the lung construct was decreased by MMP inhibition. These studies, which would not be possible using 2D cell cultures, demonstrated that NCI-H295R cells secreted active MMPs that are used for invasion in 3D. This work represents the first evidence of a 3D tumor constructs platform for ACC that can be deployed for future mechanistic studies as well as development of new targets for intervention and therapies.

A 3D adrenocortical carcinoma tumor platform for preclinical modeling of drug response and matrix metalloproteinase activity.

Adrenocortical carcinoma (ACC) has a poor prognosis, and no new drugs have been identified in decades. The absence of drug development can partly be attributed to a lack of preclinical models. Both animal models and 2D cell cultures of ACC fail to accurately mimic the disease, as animal physiology is inherently different than humans, and 2D cultures fail to represent the crucial 3D architecture. Organoids and other small 3D in vitro models of tissues or tumors can model certain complexities of human in vivo biology; however, this technology has largely yet to be applied to ACC. In this study, we describe the generation of 3D tumor constructs from an established ACC cell line, NCI-H295R. NCI-H295R cells were encapsulated to generate 3D ACC constructs. Tumor constructs were assessed for biomarker expression, viability, proliferation, and cortisol production. In addition, matrix metalloproteinase (MMP) functionality was assessed directly using fluorogenic MMP-sensitive biosensors and through infusion of NCI-H295R cells into a metastasis-on-a-chip microfluidic device platform. ACC tumor constructs showed expression of biomarkers associated with ACC, including SF-1, Melan A, and inhibin alpha. Treatment of ACC tumor constructs with chemotherapeutics demonstrated decreased drug sensitivity compared to 2D cell culture. Since most tumor cells migrate through tissue using MMPs to break down extracellular matrix, we validated the utility of ACC tumor constructs by integrating fluorogenic MMP-sensitive peptide biosensors within the tumor constructs. Lastly, in our metastasis-on-a-chip device, NCI-H295R cells successfully engrafted in a downstream lung cell line-based construct, but invasion distance into the lung construct was decreased by MMP inhibition. These studies, which would not be possible using 2D cell cultures, demonstrated that NCI-H295R cells secreted active MMPs that are used for invasion in 3D. This work represents the first evidence of a 3D tumor constructs platform for ACC that can be deployed for future mechanistic studies as well as development of new targets for intervention and therapies.

Tumor cell-conditioned media drives collagen remodeling via fibroblast and pericyte activation in an in vitro premetastatic niche model.

Primary tumors secrete large quantities of cytokines and exosomes into the bloodstream, which are uptaken at downstream sites and induce a pro-fibrotic, pro-inflammatory premetastatic niche. Niche development is associated with later increased metastatic burden, but the cellular and matrix changes in the niche that facilitate metastasis are yet unknown. Furthermore, there is no current standard model to study this phenomenon. Here, biofabricated collagen and hyaluronic acid hydrogel models were employed to identify matrix changes elicited by pericytes and fibroblasts after exposure to colorectal cancer-secreted factors. Focusing on myofibroblast activation and collagen remodeling, we report fibroblast activation and pericyte stunting in response to tumor signaling. In addition, we characterize contributions of both cell types to matrix dysregulation via collagen degradation, deposition, and architectural remodeling. With these findings, we discuss potential impacts on tissue stiffening and vascular leakiness and suggest pathways of interest for future mechanistic studies of metastatic cell-premetastatic niche interactions.

Pten regulates collagen fibrillogenesis by fibroblasts through SPARC.

Collagen deposition contributes to both high mammographic density and breast cancer progression. Low stromal PTEN expression has been observed in as many as half of breast tumors and is associated with increases in collagen deposition, however the mechanism connecting PTEN loss to increased collagen deposition remains unclear. Here, we demonstrate that Pten knockout in fibroblasts using an Fsp-Cre;PtenloxP/loxP mouse model increases collagen fiber number and fiber size within the mammary gland. Pten knockout additionally upregulated Sparc transcription in fibroblasts and promoted collagen shuttling out of the cell. Interestingly, SPARC mRNA expression was observed to be significantly elevated in the tumor stroma as compared to the normal breast in several patient cohorts. While SPARC knockdown via shRNA did not affect collagen shuttling, it notably decreased assembly of exogenous collagen. In addition, SPARC knockdown decreased fibronectin assembly and alignment of the extracellular matrix in an in vitro fibroblast-derived matrix model. Overall, these data indicate upregulation of SPARC is a mechanism by which PTEN regulates collagen deposition in the mammary gland stroma.