A simple biomechanical test with real-time displacement and strain mapping is reported, which provides displacement vectors and principal strain directions during the mechanical characterization of heart valve tissues. The maps reported in the current study allow us to quickly identify the approximate strain imposed on a location in the samples. The biomechanical results show that the aortic valves exhibit stronger anisotropic mechanical behavior than that of the pulmonary valves before 18% strain equibiaxial stretching. In contrast, the pulmonary valves exhibit stronger anisotropic mechanical behavior than aortic valves beyond 28% strain equibiaxial stretching. Simple biochemical tests are also conducted. Collagens are extracted at different time points (24, 48, 72, and 120 h) at different locations in the samples. The results show that extraction time plays an important role in determining collagen concentration, in which a minimum of 72 h of extraction is required to obtain saturated collagen concentration. This work provides an easy approach for quantifying biomechanical and biochemical properties of semilunar heart valve tissues, and potentially facilitates the development of tissue engineered heart valves.
It is well documented that the tumor microenvironment profoundly impacts the etiology and progression of breast cancer, yet the contribution of the resident microbiome within breast tissue remains poorly understood. Tumor microenvironmental conditions, such as hypoxia and dense tumor stroma, predispose progressive phenotypes and therapy resistance, however the role of bacteria in this interplay remains uncharacterized. We hypothesized that the effect of individual bacterial secreted molecules on breast cancer viability and proliferation would be modulated by these tumor-relevant stressors differentially for cells at varying stages of progression. To test this, we incubated human breast adenocarcinoma cells (MDA-MB-231, MCF-DCIS.com) and non-malignant breast epithelial cells (MCF-10A) with N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL), a quorum-sensing molecule from Pseudomonas aeruginosa that regulates bacterial stress responses. This molecule was selected because Pseudomonas was recently characterized as a significant fraction of the breast tissue microbiome and OdDHL is documented to impact mammalian cell viability. After OdDHL treatment, we demonstrated the greatest decrease in viability with the more malignant MDA-MB-231 cells and an intermediate MCF-DCIS.com (ductal carcinoma in situ) response. The responses were also culture condition (i.e. microenvironment) dependent. These results contrast the MCF-10A response, which demonstrated no change in viability in any culture condition. We further determined that the observed trends in breast cancer viability were due to modulation of proliferation for both cell types, as well as the induction of necrosis for MDA-MB-231 cells in all conditions. Our results provide evidence that bacterial quorum-sensing molecules interact with the host tissue environment to modulate breast cancer viability and proliferation, and that the effect of OdDHL is dependent on both cell type as well as microenvironment. Understanding the interactions between bacterial signaling molecules and the host tissue environment will allow for future studies that determine the contribution of bacteria to the onset, progression, and therapy response of breast cancer.
23 24 the breast tissue microbiome and OdDHL is documented to have significant effects on 36 mammalian cells. We found differences in the MDA-MB-231 and MCF-DCIS.com viability 37 after OdDHL treatment that were cell type and culture condition (i.e. microenvironment) 38 dependent. This result was in contrast to the MCF-10A cells, which demonstrated no change in 39 viability over the OdDHL concentration range examined, in any culture condition. We further 40 determined that the observed trends in breast cancer viability were due to modulation of 41 proliferation for both cell types, as well as the induction of necrosis for MDA-MB-231 cells in 42 all conditions. Our results provide evidence that bacterial quorum-sensing molecules interact 43 with the host environment to modulate breast cancer viability and proliferation, and that the 44 effect of OdDHL is dependent on both cell type as well as microenvironment. Understanding the 45 interactions between bacterial signaling molecules and the host tissue environment will allow for 46 3 future studies that determine the contribution of bacteria to the onset, progression, and therapy 47 response of breast cancer. 48 49 Introduction The tumor microenvironment is now a widely recognized and well-studied 50 contributor to cancer dynamics, particularly for breast cancer. While increased matrix density, 51 programming of cancer-associated stromal cells, evolving gradients of oxygen and nutrients, and 52 leaky vasculature have all been implicated as key players in breast cancer progression (1-4), the 53 impact of the recently identified breast tissue resident microbiotic niche has received little 54 attention. Beyond the effects of pathogenic or tumorigenic bacteria such as Chlamydophila 55 pneumonia, Salmonella typhi, Streptococcus gallolyticus (5), Helicobacter pylori (6) and 56 Fusobacterium nucleatum (7), the majority of analyses of tumor-microbiome interactions have 57 centered on local cell-cell interactions within the gut microenvironment, or more systemic 58 immune effects influenced by gut microbiota (8). Only a handful of studies have been conducted 59 to investigate the influences of tissue-resident bacteria in other tumor sites, such as for breast 60 cancer (9-11). Even fewer have investigated how small molecules released from resident bacteria 61 may interact with cells in the presence of other critical microenvironmental factors, e.g. tumor 62 hypoxia, to regulate cancer progression. In an effort to address these questions, we investigated 63 interactions between the quorum-sensing molecule N-(3-oxododecanoyl)-L-homoserine lactone 64 (OdDHL) and the breast tumor relevant microenvironmental cues of a stiff collagen-derived 65 tissue mimic and hypoxia. This representative study will aid in our understanding of how the 66 understudied breast tissue microbiome may contribute to disease phenotypes, patient-to-patient 67 variability, and cancer progression. 68 69 131 For 2D experimental conditions, cells were seeded in 48-well polystyrene plates at a 132 concentration ...
Tumoroid cultures, also known as cancer organoids, have been shown to maintain patient-specific mutational and gene expression profiles over the course of long-term culture better than traditional cancer lines. Despite the physiological relevance of tumoroid models, they have yet to supplant cancer lines, particularly for high-throughput screening (HTS) applications, in large part due to the relative difficulty of the culture workflow. The current gold standard tumoroid culture method involves embedding the cells into a scaffold (most commonly basement membrane extract or BME), which is highly manual, costly in time and resources, and difficult to implement in HTS workflows. We have developed a novel tumoroid culture medium, named GibcoTM OncoProTM Tumoroid Culture Medium, and method in which the addition of diluted BME to tumoroid suspension cultures preserves apical-in polarity (equivalent to that of embedded cultures) while leveraging the benefits of a suspension culture workflow. Additionally, histology for a subset of suspension cultures have been compared with xenograft tumors formed in mice and demonstrate that the suspension culture maintains similar cellular organization. Our system enables a single user to generate hundreds of millions of cells, a feat that would require hundreds of BME domes using standard tumoroid culture methods, and has been shown compatible with colorectal, lung, pancreatic, and head and neck tumoroid lines that were derived in embedded culture with various complete media formulations. Critically, our data confirm that, when paired with our novel tumoroid culture medium, suspension culture maintains patient-specific characteristics comparably to embedded culture when passaged side-by-side for multiple months. Tumoroid lines representing the four unique cancer indications evaluated maintained patient-specific mutational and gene expression profiles (both ≥ 90% correlated with the original material) over the course of culture. An evaluation of differentially expressed genes from a panel of over 20,000 human RefSeq genes showed that there was no change (greater than a two-fold increase/decrease; p-value < 0.05) in gene expression for ≥ 98% of genes, on average, between embedded and suspension culture. Following scale up in suspension culture, we also show that tumoroids can be plated using automated liquid handling techniques for downstream HTS assays. Taken together, our data indicate that this suspension culture workflow paired with OncoPro Tumoroid Culture Medium maintains all critical characteristics of tumoroid lines grown in the traditional embedded culture workflow, while providing the scalability and compatibility with liquid handling that will enable greater adoption of these more physiologically relevant cancer models. Citation Format: Brittany N. Balhouse, Colin Paul, Chris Yankaskas, Shyanne Salen, Sybelle Djikeng, Pradip Shahi Thakuri, Anthony Chatman, Amber Bullock, Matthew Dallas, David Kuninger. How low can you go: Maintenance of tumoroid phenotype with a highly scalable and automation-compatible reduced-ECM suspension culture method [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 158.
In vitro cancer research often fails to translate to the clinic, in part due to the use of traditional 2D cancer cell lines as models, which fail to resemble primary cancer cells by a variety of measures, including high mutational burden. An emerging solution to this problem is to replace traditional cell lines with patient tissue-derived cells expanded in 3D, also known as tumoroids or cancer organoids. We developed a defined, serum- and conditioned medium-free system, GibcoTM OncoProTM Tumoroid Culture Medium, that can be used to derive stable tumoroid lines from a variety of tissue sources and maintains the phenotype and genotype of patient-derived tumor cells. By supplementing the base medium with indication-specific growth factors, tumoroid lines were derived from colorectal, lung, and endometrial cancers from both fresh surgical resections and cryopreserved primary cancer cells. To demonstrate the utility of these patient-derived cells as long-term in vitro models, colorectal and lung tumoroid lines were derived from multiple donors and cultured for up to 50 passages. Brightfield microscopy, cell counts, and next-generation sequencing were used to assess maintenance of tumoroid morphology, growth rate, gene expression patterns, and genomic mutations. Patient-derived tumoroid cultures adopted donor-specific morphologies that were maintained during long-term culture. Cell doubling time tended to stabilize within the first few passages as cultures established, was donor-dependent, and averaged around 65 hours for colorectal tumoroids - on par with that of traditional 2D cancer cell lines - and 90-100 hours for lung and endometrial tumoroids, respectively. Importantly, tumoroid lines maintained their gene expression pattern for over 20,000 human RefSeq genes during long-term culture, with correlation between initial tumor material and late-passage samples of R>0.8. Distinct molecular subtypes of colorectal cancer were preserved in cultured tumoroids. The allelic frequency of single nucleotide variations (SNVs) in 161 highly relevant cancer genes was also highly correlated (R>0.9) between uncultured tissue and late-passage tumoroids. Within SNVs, transition/transversion mutation ratios were conserved. Tumoroids were cryopreserved and recovered during this study, demonstrating that biobanking of colorectal and lung tumoroids should not impact their long-term stability. Finally, a subset of the derived colorectal and lung tumoroids were tested and shown to be tumorigenic in mice, where subsequent histology of the tumor was similar to that of in vitro cultures. Altogether, tumoroid derivation and culture in this novel medium enables the long-term preservation of patient-specific cellular genotype and phenotype, which should allow for expansion, biobanking, and performance of experimental repeats within the same patient tissue-derived cultures across labs and over time. Citation Format: Chris Yankaskas, Brittany Balhouse, Colin Paul, Shyanne Salen, Sybelle Djikeng, Pradip Shahi Thakuri, Mark Kennedy, Matt Dallas, David Kuninger. Derivation and long-term maintenance of patient-derived tumoroid lines in a defined, serum-free medium [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 160.
Tumoroid technology enables culture of patient tissue-derived cancer cells in 3D, with retention of key characteristics from the original patient tumor. However, current tumoroid culture relies on labor-intensive media formulations and culture workflows. To address these issues, we have developed the serum- and conditioned medium-free Gibco™ OncoPro™ Tumoroid Culture Medium to enable derivation and expansion of patient-derived lines from multiple cancer indications. The system has been optimized to retain key patient genotypic and phenotypic characteristics during in vitro culture in a Wnt agonist-free system that can be easily adopted and transferred between labs. To test the applicability of our system with previously established tumoroid/cancer organoid models, we procured publicly available cancer models from the National Cancer Institute Patient-Derived Models Repository (NCI PDMR). Multiple colorectal, lung, pancreas, and head and neck tumoroid models were tested, including tumoroids originally derived in conditioned medium containing Wnt-3A, R-spondin 3, and Noggin. Cells were assessed for survival, growth, and fidelity to starting material in both OncoPro Tumoroid Culture Medium and in PDMR-recommended homebrew media. Growth and morphology of tumoroid cells was monitored and was comparable between culture conditions. To test for genetic stability of cultures over time, the mutational status of organoids expanded in each condition was characterized using targeted NGS. Both SNV allelic frequency and ploidy values were conserved from initial starting material following expansion to cryopreservation-competent banks (>10e6 cells). Gene expression levels across over >20,000 human RefSeq genes were compared between culture methods and showed high (>0.9) correlation, and Wnt-related signaling pathways were not differentially regulated between media types. In select cases, tumoroid cells expanded in each condition were also analyzed by flow cytometry using optimized antibody panels and protocols. Total cell viability and expression of EpCAM, CD45, CD31, smooth muscle actin, vimentin, CDX2, CEACAM, and cytokeratin 7 was nearly identical across culture conditions. Taken together, our approach represents a simple and effective method to expand and maintain patient-derived human cancer organoids in vitro, with no clear adverse effects from moving to a Wnt-free system. Citation Format: Colin Paul, Brittany Balhouse, Chris Yankaskas, Shyanne Salen, Sybelle Djikeng, Pradip Shahi Thakuri, Matt Dallas, David Kuninger. Expansion of established patient-derived tumoroids in a novel serum-free, Wnt-free media system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 177.
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