Integrated computational and experimental pipeline for quantifying local cell-matrix interactions.

Cellular interactions with the extracellular matrix (ECM) play a key role in modulating biological processes. While studies have identified key molecular factors of these interactions, the mechanical regulation associated with these interactions is not well characterized. To address this, we present an image analysis platform to analyze time-dependent dynamics observed in lung fibroblasts embedded in a 3D collagen matrix. Combining drug studies with quantitative analysis of cell-matrix interactions, our results are able to provide cellular level quantitative insights for mechanical and biophysical phenomena relevant to cell-ECM interactions. This system overall represents an initial pipeline for understanding cell mechanics in a 3D collagen gel and their implications in a physiologically relevant context.

Decellularized mice colons as models to study the contribution of the extracellular matrix to cell behavior and colon cancer progression.

Current 3D culture models to study colorectal cancer lack architectural support and signaling proteins provided by the tissue extracellular matrix (ECM) which may influence cell behavior and cancer progression. Therefore, the ability to study cancer cells in the context of a matrix that is physiologically more relevant and to understand how the ECM affects cancer progression has been understudied. To address this, we developed an ex-vivo 3D system, provided by intact wild type (WT) and colon cancer susceptible decellularized mouse colons (DMC), to support the growth of human cancer cells. DMC are free of viable cells but still contain extracellular matrix proteins including subsets of collagens. Stiffness, an important mechanical property, is also maintained in DMCs. Importantly, we observed that the DMC is permissive for cell proliferation and differentiation of a human colon cancer cell line (HT-29). Notably, the ability of cells in the WT DMC to differentiate was also greater when compared to Matrigel™, an extracellular matrix extract from a mouse tumor cell line. Additionally, we observed in invasion assays that DMC obtained from polyps from a colon cancer susceptible mouse model facilitated increased cell migration/invasion of colorectal cancer cells and immortalized non-tumor colonic epithelial cells compared to DMC from WT mice. Finally, using mass spectrometry, we identified extracellular matrix proteins that are more abundant in DMC from a colorectal cancer mouse model compared to age and sex-matched WT mice. We propose that these abundantly expressed proteins in the tumor microenvironment are potentially involved in colorectal cancer progression. STATEMENT OF SIGNIFICANCE: Decellularized matrices, when properly produced, are attractive biomaterials for tissue regeneration and replacement. We show here that the mouse decellularized matrices can also be repurposed to elucidate how the extracellular matrix influences human cell behavior and cancer progression. To do this we produce decellularized matrices, from mice colonic tissue, that have preserved tissue mechanical and structural properties. We demonstrate that the matrix better supports the differentiation of HT-29 cells, a colonic cancer cell line, compared to Matrigel™. Additionally, we show that the extracellular matrix contributes to colon cancer progression via invasion assays using extracellular matrix extracts. Finally, we use mass spectrometry to identify ECM proteins that are more abundant in colonic polyps compared to adjacent tissue regions. This model system may have therapeutic implications for colorectal cancer patients.

Analysis of Keloid Response to 5-Fluorouracil Treatment and Long-Term Prevention of Keloid Recurrence.

Keloids are benign fibroproliferative skin tumors that can cause disfigurement and disability. Although they frequently recur after excision or medical management and can affect 6 to 16 percent of African Americans, there is no gold standard therapy. Keloids are challenging to study because there are no animal or in vitro models of this disorder. This makes it very difficult to validate data from treated tissue samples or cells and develop targeted therapies for this disease. In this study, the authors demonstrate that intralesional 5-fluorouracil injection after keloid excision prevents recurrence for 2 years, with no reported adverse events. The authors analyze the expression of treated and untreated biopsy specimens of the same keloids in their native context to capture insights that may be missed by in vitro cell culture models and correct for intrakeloid variability. Random forest analysis of the microarray data dramatically increased the statistical power of the authors' results, permitting hypothesis-free creation of a gene expression profile of 5-fluorouracil-treated keloids. Through this analysis, the authors found a set of genes, including YAP1 and CCL-2, whose expression changes predict 5-fluorouracil therapy status and include genes that have not previously been associated with keloid biology and are of unknown function. The authors further describe keloid heterogeneity for the first time using multidimensional analysis of their microarray results. The methods and tools the authors developed in this research may overcome some of the challenges in studying keloids and developing effective treatments for this disease. CLINICAL QUESTION/LEVEL OF EVIDENCE:: Therapeutic, V.

Neuronal calcium sensor 1 (NCS1) promotes motility and metastatic spread of breast cancer cells in vitro and in vivo.

Increased levels of the calcium-binding protein neuronal calcium sensor 1 (NCS1) predict an unfavorable patient outcome in several aggressive cancers, including breast and liver tumors. Previous studies suggest that NCS1 overexpression facilitates metastatic spread of these cancers. To investigate this hypothesis, we explored the effects of NCS1 overexpression on cell proliferation, survival, and migration patterns in vitro in 2- and 3-dimensional (2/3-D). Furthermore, we translated our results into an in vivo mouse xenograft model. Cell-based proliferation assays were used to demonstrate the effects of overexpression of NCS1 on growth rates. In vitro colony formation and wound healing experiments were performed and 3-D migration dynamics were studied using collagen gels. Nude mice were injected with breast cancer cells to monitor NCS1-dependent metastasis formation over time. We observed that increased NCS1 levels do not change cellular growth rates, but do significantly increase 2- and 3-D migration dynamics in vitro. Likewise, NCS1-overexpressing cells have an increased capacity to form distant metastases and demonstrate better survival and less necrosis in vivo. We found that NCS1 preferentially localizes to the leading edge of cells and overexpression increases the motility of cancer cells. Furthermore, this phenotype is correlated with an increased number of metastases in a xenograft model. These results lay the foundation for exploring the relevance of an NCS1-mediated pathway as a metastatic biomarker and as a target for pharmacologic interventions.-Apasu, J. E., Schuette, D., LaRanger, R., Steinle, J. A., Nguyen, L. D., Grosshans, H. K., Zhang, M., Cai, W. L., Yan, Q., Robert, M. E., Mak, M., Ehrlich, B. E. Neuronal calcium sensor 1 (NCS1) promotes motility and metastatic spread of breast cancer cells in vitro and in vivo.

Telomerase-Mediated Strategy for Overcoming Non-Small Cell Lung Cancer Targeted Therapy and Chemotherapy Resistance.

Standard and targeted cancer therapies for late-stage cancer patients almost universally fail due to tumor heterogeneity/plasticity and intrinsic or acquired drug resistance. We used the telomerase substrate nucleoside precursor, 6-thio-2'-deoxyguanosine (6-thio-dG), to target telomerase-expressing non-small cell lung cancer cells resistant to EGFR-inhibitors and commonly used chemotherapy combinations. Colony formation assays, human xenografts as well as syngeneic and genetically engineered immune competent mouse models of lung cancer were used to test the effect of 6-thio-dG on targeted therapy- and chemotherapy-resistant lung cancer human cells and mouse models. We observed that erlotinib-, paclitaxel/carboplatin-, and gemcitabine/cisplatin-resistant cells were highly sensitive to 6-thio-dG in cell culture and in mouse models. 6-thio-dG, with a known mechanism of action, is a potential novel therapeutic approach to prolong disease control of therapy-resistant lung cancer patients with minimal toxicities.

Long-term culture and cloning of primary human bronchial basal cells that maintain multipotent differentiation capacity and CFTR channel function.

While primary cystic fibrosis (CF) and non-CF human bronchial epithelial basal cells (HBECs) accurately represent in vivo phenotypes, one barrier to their wider use has been a limited ability to clone and expand cells in sufficient numbers to produce rare genotypes using genome-editing tools. Recently, conditional reprogramming of cells (CRC) with a Rho-associated protein kinase (ROCK) inhibitor and culture on an irradiated fibroblast feeder layer resulted in extension of the life span of HBECs, but differentiation capacity and CF transmembrane conductance regulator (CFTR) function decreased as a function of passage. This report details modifications to the standard HBEC CRC protocol (Mod CRC), including the use of bronchial epithelial cell growth medium, instead of F medium, and 2% O2, instead of 21% O2, that extend HBEC life span while preserving multipotent differentiation capacity and CFTR function. Critically, Mod CRC conditions support clonal growth of primary HBECs from a single cell, and the resulting clonal HBEC population maintains multipotent differentiation capacity, including CFTR function, permitting gene editing of these cells. As a proof-of-concept, CRISPR/Cas9 genome editing and cloning were used to introduce insertions/deletions in CFTR exon 11. Mod CRC conditions overcome many barriers to the expanded use of HBECs for basic research and drug screens. Importantly, Mod CRC conditions support the creation of isogenic cell lines in which CFTR is mutant or wild-type in the same genetic background with no history of CF to enable determination of the primary defects of mutant CFTR.

Reconstituting Mouse Lungs with Conditionally Reprogrammed Human Bronchial Epithelial Cells.

We developed methods for conditionally reprogramming (CR) primary human bronchial epithelial cells (HBECs) to extend their functional lifespan and permit their differentiation into both upper and lower airway lung epithelium. We also developed a bioreactor to support vascular perfusion and rhythmic breathing of decellularized mouse lungs reconstituted with CR HBECs isolated from patients with and without cystic fibrosis (CF). While conditionally reprogrammed cells only differentiate into an upper airway epithelium after 35 days at the air-liquid interface, in reconstituted lungs these cells differentiate into upper airway bronchial epithelium and lower airway alveolar structures after 12 days. Rapid scale-up and the ability to obtain clonal derivatives of primary patient-derived HBECs without the need for genetic manipulation may permit rapid reconstitution of the lung epithelium; facilitating the study of lung disease in tissue-engineered models.

Comparison of DNA Quantification Methods for Next Generation Sequencing.

Next Generation Sequencing (NGS) is a powerful tool that depends on loading a precise amount of DNA onto a flowcell. NGS strategies have expanded our ability to investigate genomic phenomena by referencing mutations in cancer and diseases through large-scale genotyping, developing methods to map rare chromatin interactions (4C; 5C and Hi-C) and identifying chromatin features associated with regulatory elements (ChIP-seq, Bis-Seq, ChiA-PET). While many methods are available for DNA library quantification, there is no unambiguous gold standard. Most techniques use PCR to amplify DNA libraries to obtain sufficient quantities for optical density measurement. However, increased PCR cycles can distort the library's heterogeneity and prevent the detection of rare variants. In this analysis, we compared new digital PCR technologies (droplet digital PCR; ddPCR, ddPCR-Tail) with standard methods for the titration of NGS libraries. DdPCR-Tail is comparable to qPCR and fluorometry (QuBit) and allows sensitive quantification by analysis of barcode repartition after sequencing of multiplexed samples. This study provides a direct comparison between quantification methods throughout a complete sequencing experiment and provides the impetus to use ddPCR-based quantification for improvement of NGS quality.