Circulating DNA (ctDNA) is acknowledged as a potential diagnostic tool for various cancers including colorectal cancer, especially when considering the detection of mutations. Certainly due to lack ...of normalization of the experimental conditions, previous reports present many discrepancies and contradictory data on the analysis of the concentration of total ctDNA and on the proportion of tumour-derived ctDNA fragments.
In order to rigorously analyse ctDNA, we thoroughly investigated ctDNA size distribution. We used a highly specific Q-PCR assay and athymic nude mice xenografted with SW620 or HT29 human colon cancer cells, and we correlated our results by examining plasma from metastatic CRC patients.
Fragmentation and concentration of tumour-derived ctDNA is positively correlated with tumour weight. CtDNA quantification by Q-PCR depends on the amplified target length and is optimal for 60-100 bp fragments. Q-PCR analysis of plasma samples from xenografted mice and cancer patients showed that tumour-derived ctDNA exhibits a specific amount profile based on ctDNA size and significant higher ctDNA fragmentation. Metastatic colorectal patients (n = 12) showed nearly 5-fold higher mean ctDNA fragmentation than healthy individuals (n = 16).
The extracellular matrix (ECM) of tissues is an important mediator of cell function. Moreover, understanding cellular dynamics within their specific tissue context is also important for developmental ...biology, cancer research, and regenerative medicine. However, robust in vitro models that incorporate tissue-specific microenvironments are lacking. Here we describe a novel mammary-specific culture protocol that combines a self-gelling hydrogel comprised solely of ECM from decellularized rat or human breast tissue with the use of our previously described 3D bioprinting platform. We initially demonstrate that undigested and decellularized mammary tissue can support mammary epithelial and tumor cell growth. We then describe a methodology for generating mammary ECM extracts that can spontaneously gel to form hydrogels. These ECM hydrogels retain unique structural and signaling profiles that elicit differential responses when normal mammary and breast cancer cells are cultured within them. Using our bioprinter, we establish that we can generate large organoids/tumoroids in the all mammary-derived hydrogel. These findings demonstrate that our system allows for growth of organoids/tumoroids in a tissue-specific matrix with unique properties, thus providing a suitable platform for ECM and epithelial/cancer cell studies. STATEMENT OF SIGNIFICANCE: Factors within extracellular matrices (ECMs) are specific to their tissue of origin. It has been shown that tissue specific factors within the mammary gland's ECM have pronounced effects on cellular differentiation and cancer behavior. Understanding the role of the ECM in controlling cell fate has major implications for developmental biology, tissue engineering, and cancer therapy. However, in vitro models to study cellular interactions with tissue specific ECM are lacking. Here we describe the generation of 3D hydrogels consisting solely of human or mouse mammary ECM. We demonstrate that these novel 3D culture substrates can sustain large 3D bioprinted organoid and tumoroid formation. This is the first demonstration of an all mammary ECM culture system capable of sustaining large structural growths.
AXL receptor tyrosine kinase has been described as a relevant molecular marker and a key player in invasiveness, especially in triple-negative breast cancer (TNBC).
We evaluate the antitumor efficacy ...of the anti-AXL monoclonal antibody 20G7-D9 in several TNBC cell xenografts or patient-derived xenograft (PDX) models and decipher the underlying mechanisms. In a dataset of 254 basal-like breast cancer samples, genes correlated with
expression are enriched in EMT, migration, and invasion signaling pathways.
Treatment with 20G7-D9 inhibited tumor growth and bone metastasis formation in AXL-positive TNBC cell xenografts or PDX, but not in AXL-negative PDX, highlighting AXL role in cancer growth and invasion.
stimulation of AXL-positive cancer cells by its ligand GAS6 induced the expression of several EMT-associated genes (
, and
) through an intracellular signaling implicating the transcription factor FRA-1, important in cell invasion and plasticity, and increased their migration/invasion capacity. 20G7-D9 induced AXL degradation and inhibited all AXL/GAS6-dependent cell signaling implicated in EMT and in cell migration/invasion.
The anti-AXL antibody 20G7-D9 represents a promising therapeutic strategy in TNBC with mesenchymal features by inhibiting AXL-dependent EMT, tumor growth, and metastasis formation.
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Standard three-dimensional (3D) in vitro culture techniques, such as those used for mammary epithelial cells, rely on random distribution of cells within hydrogels. Although these systems offer ...advantages over traditional 2D models, limitations persist owing to the lack of control over cellular placement within the hydrogel. This results in experimental inconsistencies and random organoid morphology. Robust, high-throughput experimentation requires greater standardization of 3D epithelial culture techniques.
Here, we detail the use of a 3D bioprinting platform as an investigative tool to control the 3D formation of organoids through the "self-assembly" of human mammary epithelial cells. Experimental bioprinting procedures were optimized to enable the formation of controlled arrays of individual mammary organoids. We define the distance and cell number parameters necessary to print individual organoids that do not interact between print locations as well as those required to generate large contiguous organoids connected through multiple print locations.
We demonstrate that as few as 10 cells can be used to form 3D mammary structures in a single print and that prints up to 500 μm apart can fuse to form single large structures. Using these fusion parameters, we demonstrate that both linear and non-linear (contiguous circles) can be generated with sizes of 3 mm in length/diameter. We confirm that cells from individual prints interact to form structures with a contiguous lumen. Finally, we demonstrate that organoids can be printed into human collagen hydrogels, allowing for all-human 3D culture systems.
Our platform is adaptable to different culturing protocols and is superior to traditional random 3D culture techniques in efficiency, reproducibility, and scalability. Importantly, owing to the low-cost accessibility and computer numerical control-driven platform of our 3D bioprinter, we have the ability to disseminate our experiments with absolute precision to interested laboratories.
Carotenoids are among the most important organic compounds present in Nature and play several essential roles in biology. Their configuration is responsible for their specific photophysical ...properties, which can be tailored by changes in their molecular structure and in the surrounding environment. In this review, we give a general description of the main electronic and vibrational properties of carotenoids. In the first part, we describe how the electronic and vibrational properties are related to the molecular configuration of carotenoids. We show how modifications to their configuration, as well as the addition of functional groups, can affect the length of the conjugated chain. We describe the concept of effective conjugation length, and its relationship to the S0 → S2 electronic transition, the decay rate of the S1 energetic level and the frequency of the ν1 Raman band. We then consider the dependence of these properties on extrinsic parameters such as the polarizability of their environment, and how this information (S0 → S2 electronic transition, ν1 band position, effective conjugation length and polarizability of the environment) can be represented on a single graph. In the second part of the review, we use a number of specific examples to show that the relationships can be used to disentangle the different mechanisms tuning the functional properties of protein-bound carotenoids.
CYP17 inhibitors for prostate cancer therapy Vasaitis, Tadas S.; Bruno, Robert D.; Njar, Vincent C.O.
Journal of steroid biochemistry and molecular biology/The Journal of steroid biochemistry and molecular biology,
05/2011, Volume:
125, Issue:
1
Journal Article
Peer reviewed
Open access
▶ We reviewed the impact of androgen in castration resistant prostate cancer (CRPC). ▶ Benefits to prostate cancer patients is observed following inhibition of CYP17 enzyme. ▶ CYP17 is a prime target ...for development of new drugs to treat prostate cancer. ▶ We highlight advanced clinical evaluations of ketoconazole and abiraterone acetate. ▶ We also highlight early clinical evaluations of VN/124-1 (TOK-001) and TAK-700.
Prostate cancer (PC) is now the second most prevalent cause of death in men in the USA and Europe. At present, the major treatment options include surgical or medical castration. These strategies cause ablation of the production of testosterone (T), dihydrotestosterone (DHT) and related androgens by the testes. However, because these procedures do not affect adrenal, prostate and other tissues’ androgen production, they are often combined with androgen receptor antagonists to block their action. Indeed, recent studies have unequivocally established that in castration-resistant prostate cancer (CRPC) many androgen-regulated genes become re-expressed and tissue androgen levels increase despite low serum levels.
Clearly, inhibition of the key enzyme which catalyzes the biosynthesis of androgens from pregnane precursors, 17α-hydroxy/17,20-lyase (hereafter referred to as CYP17) could prevent androgen production from all sources. Thus, total ablation of androgen production by potent CYP17 inhibitors may provide effective treatment of prostate cancer patients. This review highlights the role of androgen biosynthesis in the progression of prostate cancer and the impact of CYP17 inhibitors, such as ketoconazole, abiraterone acetate, VN/124-1 (TOK-001) and TAK-700 in the clinic and in clinical development.
Article from the special issue on Targeted Inhibitors.
The normal mammary microenvironment can suppress tumorigenesis and redirect cancer cells to adopt a normal mammary epithelial cell fate in vivo. Understanding of this phenomenon offers great promise ...for novel treatment and detection strategies in cancer, but current model systems make mechanistic insights into the process difficult. We have recently described a low-cost bioprinting platform designed to be accessible for basic cell biology laboratories. Here we report the use of this system for the study of tumorigenesis and microenvironmental redirection of breast cancer cells. We show our bioprinter significantly increases tumoroid formation in 3D collagen gels and allows for precise generation of tumoroid arrays. We also demonstrate that we can mimic published in vivo findings by co-printing cancer cells along with normal mammary epithelial cells to generate chimeric organoids. These chimeric organoids contain cancer cells that take part in normal luminal formation. Furthermore, we show for the first time that cancer cells within chimeric structures have a significant increase in 5-hydroxymethylcytosine levels as compared to bioprinted tumoroids. These results demonstrate the capacity of our 3D bioprinting platform to study tumorigenesis and microenvironmental control of breast cancer and highlight a novel mechanistic insight into the process of microenvironmental control of cancer.
Cytochrome P450s (CYPs) represent a large class of heme-containing enzymes that catalyze the metabolism of multitudes of substrates both endogenous and exogenous. Until recently, however, CYPs have ...been largely overlooked in cancer drug development, acknowledged only for their role in phase I metabolism of chemotherapeutics. The first successful strategy targeting CYP enzymes in cancer therapy was the development of potent inhibitors of CYP19 (aromatase) for the treatment of breast cancer. Aromatase inhibitors ushered in a new era in hormone ablation therapy for estrogen dependent cancers, and have paved the way for similar strategies (i.e., inhibition of CYP17) that combat androgen dependent prostate cancer. Identification of CYPs involved in the inactivation of anti-cancer metabolites of vitamin D
3 and vitamin A has triggered development of agents that target these enzymes as well. The discovery of the over-expression of exogenous metabolizing CYPs, such as CYP1B1, in cancer cells has roused interest in the development of inhibitors for chemoprevention and of prodrugs designed to be activated by CYPs only in cancer cells. Finally, the expression of CYPs within tumors has been utilized in the development of bioreductive molecules that are activated by CYPs only under hypoxic conditions. This review offers the first comprehensive analysis of strategies in drug development that either inhibit or exploit CYP enzymes for the treatment of cancer.
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