Multi compartmental 3D breast cancer disease model–recapitulating tumor complexity in in-vitro

Breast cancer is the most common ailment among women. In 2020, it had the highest incidence of any type of cancer. Many Phase II and III anti-cancer drugs fail due to efficacy, durability, and side effects. Thus, accelerated drug screening models must be accurate. In-vivo models have been used for a long time, but delays, inconsistent results, and a greater sense of responsibility among scientists toward wildlife have led to the search for in-vitro alternatives. Stromal components support breast cancer growth and survival. Multi-compartment Transwell models may be handy instruments. Co-culturing breast cancer cells with endothelium and fibroblasts improves modelling. The extracellular matrix (ECM) supports native 3D hydrogels in natural and polymeric forms. 3D Transwell cultured tumor spheroids mimicked in-vivo pathological conditions. Tumor invasion, migration, Trans-endothelial migration, angiogenesis, and spread are studied using comprehensive models. Transwell models can create a cancer niche and conduct high-throughput drug screening, promising future applications. Our comprehensive shows how 3D in-vitro multi compartmental models may be useful in producing breast cancer stroma in Transwell culture.     

1,25-dihydroxyvitamin D3 specifically binds to a human breast cancer cell line (T47D) and stimulates growth

The T47D human breast cancer cell line contains a specific binding protein for 1.25-(OH)₂D₃, with 15000 sites per cell. The Kd (1.1 × 10^?10 M) and sedimentation coefficient on sucrose gradients (3.7S) are the same as those reported for the 1,25-(OH)₂D₃ receptor in other tissues. Other vitamin D₃ metabolites bound to the receptor with an order of affinities 1,25-(OH)₂D₃ > 1,24,25-(OH)₃D₃ > 25-OHD₃ > 24,25-(OH)₂D₃ > D₃. A new analogue 1β,25-(OH)₂D₃ was only as effective as 24,25-(OH)₂D₃ at displacing the hormone from the receptor. Cell growth was stimulated in a dose dependent manner by the addition of 1,25-(OH)₂D₃ (up to 0.8 nM) to the medium. A higher concentration of hormone was without effect.     

Killing hypoxic cell populations in a 3D tumor model with EtNBS-PDT

An outstanding problem in cancer therapy is the battle against treatment-resistant disease. This is especially true for ovarian cancer, where the majority of patients eventually succumb to treatment-resistant metastatic carcinomatosis. Limited perfusion and diffusion, acidosis, and hypoxia play major roles in the development of resistance to the majority of front-line therapeutic regimens. To overcome these limitations and eliminate otherwise spared cancer cells, we utilized the cationic photosensitizer EtNBS to treat hypoxic regions deep inside in vitro 3D models of metastatic ovarian cancer. Unlike standard regimens that fail to penetrate beyond ～150 μm, EtNBS was found to not only penetrate throughout the entirety of large (>200 μm) avascular nodules, but also concentrate into the nodules' acidic and hypoxic cores. Photodynamic therapy with EtNBS was observed to be highly effective against these hypoxic regions even at low therapeutic doses, and was capable of destroying both normoxic and hypoxic regions at higher treatment levels. Imaging studies utilizing multiphoton and confocal microscopies, as well as time-lapse optical coherence tomography (TL-OCT), revealed an inside-out pattern of cell death, with apoptosis being the primary mechanism of cell killing. Critically, EtNBS-based photodynamic therapy was found to be effective against the model tumor nodules even under severe hypoxia. The inherent ability of EtNBS photodynamic therapy to impart cytotoxicity across a wide range of tumoral oxygenation levels indicates its potential to eliminate treatment-resistant cell populations.     

A prognostic gene expression profile that predicts circulating tumor cell presence in breast cancer patients

The detection of circulating tumor cells (CTCs) in the peripheral blood and microarray gene expression profiling of the primary tumor are two promising new technologies able to provide valuable prognostic data for patients with breast cancer. Meta-analyses of several established prognostic breast cancer gene expression profiles in large patient cohorts have demonstrated that despite sharing few genes, their delineation of patients into "good prognosis" or "poor prognosis" are frequently very highly correlated, and combining prognostic profiles does not increase prognostic power. In the current study, we aimed to develop a novel profile which provided independent prognostic data by building a signature predictive of CTC status rather than outcome. Microarray gene expression data from an initial training cohort of 72 breast cancer patients for which CTC status had been determined in a previous study using a multimarker QPCR-based assay was used to develop a CTC-predictive profile. The generated profile was validated in two independent datasets of 49 and 123 patients and confirmed to be both predictive of CTC status, and independently prognostic. Importantly, the "CTC profile" also provided prognostic information independent of the well-established and powerful '70-gene' prognostic breast cancer signature. This profile therefore has the potential to not only add prognostic information to currently-available microarray tests but in some circumstances even replace blood-based prognostic CTC tests at time of diagnosis for those patients already undergoing testing by multigene assays.     

Identifying cell and molecular stress after radiation in a three-dimensional (3-D) model of oral mucositis

Mature adipocytes are excellent candidates to influence tumor behavior through heterotypic signaling processes since these cells produce hormones, growth factors, cytokines and other molecules, a heterogeneous group of molecules named adipokines. Using a 2D coculture system, we demonstrate that breast tumor cells previously co-cultivated with mature adipocytes exhibit radioresistance and an earlier and higher increase in the effector kinase Chk1, a phenotype that was associated with decreased cell death as compared to tumor cells grown alone. Interestingly, the adipocytes-induced tumor changes taking place during the coculture time preceding the exposure to IR were sufficient to confer the radioresistant effect. Notorious among the changes brought by adipocytes was the significant increase of IL-6 expression in tumor cells, whose activity may well account for the observed tumor cell protection from IR toxicity. Indeed, our data confirmed the protective role of this cytokine as tumor cells incubated after irradiation with recombinant IL-6 exhibit an increased in Chk1 phosphorylation and a radioresistant phenotype, thus far recapitulating the effects observed in the presence of adipocytes. Our current study sheds light on a new role of tumor-surrounding adipocytes in fostering a radioresistant phenotype in breast tumors, a finding that might have important clinical implications in obese patients that frequently exhibit aggressive diseases.     

Identifying cell and molecular stress after radiation in a three-dimensional (3-D) model of oral mucositis

Mucositis is a debilitating adverse effect of chemotherapy and radiation treatment. It is important to develop a simple and reliable in vitro model, which can routinely be used to screen new drugs for prevention and treatment of mucositis. Furthermore, identifying cell and molecular stresses especially in the initiation phase of mucositis in this model will help towards this end. We evaluated a three-dimensional (3-D) human oral cell culture that consisted of oral keratinocytes and fibroblasts as a model of oral mucositis. The 3-D cell culture model was irradiated with 12 or 2 Gy. Six hours after the irradiation we evaluated microscopic sections of the cell culture for evidence of morphologic changes including apoptosis. We used microarrays to compare the expression of several genes from the irradiated tissue with identical genes from tissue that was not irradiated. We found that irradiation with 12 Gy induced significant histopathologic effects including cellular apoptosis. Irradiation significantly affected the expression of several genes of the NF-kB pathway and several inflammatory cytokines, such as IL-1B, 1L-8, NF-kB1, and FOS compared to tissue that was not irradiated. We identified significant upregulation of several genes that belong to damage-associated molecular patterns (DAMPs) such as HMB1, S100A13, SA10014, and SA10016 in the 3-D tissues that received 12 Gy but not in tissues that received 2 Gy. In conclusion, this model quantifies radiation damage and this is an important first step towards the development 3-D tissue as a screening tool.     

A study of electrochemical biosensor for analysis of three-dimensional (3D) cell culture

The miniaturization of electrochemical sensors allows for the minimally invasive and cost effective examination of cellular responses at a high efficacy rate. In this work, an ink-jet printed superoxide dismutase electrode was designed, characterized, and utilized as a novel microfluidic device to examine the metabolic response of a 2D layer of macrophage cells. Since superoxide production is one of the first indicators of oxidative burst, macrophage cells were exposed within the microfluidic device to phorbol myristate acetate (PMA), a known promoter of oxidative burst, and the production of superoxide was measured. A 46 ± 19% increase in current was measured over a 30 min time period demonstrating successful detection of sustained macrophage oxidative burst, which corresponds to an increase in the superoxide production rate by 9 ± 3 attomoles/cell/s. Linear sweep voltammetry was utilized to show the selectivity of this sensor for superoxide over hydrogen peroxide. This novel controllable microfluidic system can be used to study the impact of multiple effectors from a large number of bacteria or other invaders along a 2D layer of macrophages, providing an in vitro platform for improved electrochemical studies of metabolic responses.     

Hormone receptor status, erbB2 expression and cancer stem cell characteristics of circulating tumor cells in breast cancer patients

The most important predictor for disease-free and overall survival of breast cancer patients is the presence of axillary lymph node metastasis. For surveillance during recurrence-free follow-up or in metastatic disease no marker is available at the moment. Several trials have shown the prognostic relevance of circulating tumor cells (CTC) in early and metastatic breast cancers. Indeed, only CTC that exhibit specific molecular characteristics including stem cell characteristics, could be able to create new metastasis. Hormone therapy or anti-erbB2 therapies are prescribed according to the hormone (ERα/PR expression) and erbB2 status of the initial tumor. Nonetheless, it appears that the CTC, and consequently the metastatic cells, may have a very different hormone and erbB2 status. An optimal individualized treatment could then be obtained by characterizing ERα and erbB2 status in the CTC and comparing it to the primary tumor.     

Influence of adjuvant radiotherapy on circulating epithelial tumor cells and circulating cancer stem cells in primary non-metastatic breast cancer

Background: There is an unmet need to identify biomarkers that directly reflect response to adjuvant radiotherapy (RT). Circulating epithelial tumor cells (CETCs) represent the liquid component of solid tumors and are responsible for metastatic relapse. CETC subsets with cancer stem cell characteristics, circulating cancer stem cells (cCSCs), play a pivotal role in the metastatic cascade. Monitoring the most aggressive subpopulation of CETCs could reflect the aggressiveness of the remaining tumor burden. There is limited data on the detection and monitoring changes in CETC and cCSC numbers during RT in early breast cancer.Methods: CETC numbers were analyzed prior to, at midterm and at the end of RT in 52 primary non-metastatic breast cancer patients. Hormone receptor status was determined in CETCs prior to and at the end of RT. For the identification of cCSCs cell suspensions from the peripheral blood of patients were cultured in vitro under conditions favoring growth of tumorspheres.Results: Hormone receptor status in CETCs before RT was comparable to that in primary tumor tissue. Prior to RT numbers of CETCs correlated with aggressiveness of primary tumors. cCSCs could be successfully identified and monitored during RT. Prior to RT patients treated with neoadjuvant chemotherapy had significantly higher numbers of CETCs and tumorspheres compared to patients after adjuvant chemotherapy. During RT, the number of CETCs decreased continuously in patients after neoadjuvant chemotherapy but not after adjuvant chemotherapy.Conclusion: Monitoring the number of CETCs and the CETC subset with cancer stem cell properties during RT may provide additional clinically useful prognostic information.     

Efficient tumor cell lysis mediated by a Bcl-X(L) specific T cell clone isolated from a breast cancer patient

Based on the detection of spontaneous immune responses in cancer patients with cancer of different origin, Bcl-X(L) was recently described as a highly interesting tumor antigen recognized by CD8 positive cytotoxic T lymphocytes. To further characterize Bcl-X(L) as a tumor antigen we isolated and expanded Bcl-X(L) specific T cells from the peripheral blood of a breast cancer patient hosting a strong Bcl-X(L) specific T cell response. We describe that HLA-A2 restricted Bcl-X(L) specific T cell clones very efficiently lyse peptide pulsed T2 cells. Furthermore, tumor cell lines of different origin, i.e., breast cancer, colon cancer, and melanoma, are efficiently lysed in an HLA-dependent manner. Finally, ex vivo-isolated leukemia cells, but not non-malignant B and T cells are killed by Bcl-X(L) specific T cells. Our data underline Bcl-X(L) as an universal tumor antigen widely applicable in specific anticancer immunotherapy.     

The promise of circulating tumor cell analysis in cancer management

Enumeration and molecular characterization of circulating tumor cells isolated from peripheral blood of patients with cancer can aid selection of targeted therapy for patients, monitoring of response to therapies and optimization of drug development, while also providing valuable information about intratumoral heterogeneity.     

An approach to the proteomic analysis of a breast cancer cell line (SKBR-3)

This report describes the profiling of proteins in a sample prepared by laser capture microdissection (LCM) from a breast cancer cell line (SKBR-3). This experimental approach serves as a model system for proteomic studies on selected tissue samples and for studies of specific cell types. The captured cells were isolated in a dehydrated and reduced state and solubilized with a denaturing buffer. After dilution the protein mixture was digested with trypsin and the resulting peptide mixture was fractionated by reversed phase HPLC (RPLC) and analyzed on an ion trap mass spectrometer. A key part of this study is the combination of the LCM process with an extraction/digestion procedure that allowed effective solubilization of a significant part of the cellular sample in a single step. The identity of the peptides was determined by tandem mass spectrometry measurements in which the resulting spectra were compared with genomic and proteomic databases and protein identifications were made. While only peptides with a high probability assignment were used, the interpretation of mass spectral fragmentation patterns were also confirmed by manual interpretation of the spectra. Also, for the more abundant proteins the initial protein assignment from the best match peptide was strengthened by the observation of additional confirmatory peptide identifications. Another selection criteria was correlation of the mass spectrometric studies with clinical and genomic studies of potential cancer markers in tumor samples. This proteomic study allowed identification of the following proteins: human receptor protein kinase HER-2 or ERBB-2 and related kinases HER-3 and HER-4, the gene products from breast cancer type I and II susceptibility genes and cytoskeletal components such as cytokeratins 8, 18 and 19. Other proteins include fibroblast growth factor receptor variants (FGFR-2&4) and T-lymphoma invasion and metastasis inducing protein 1 (TIAM1). In addition several nonreceptor protein kinases YES, FAK and JAK-1 and 3 were identified. Since the study was performed on a limited number of cells (approximately 10,000) it raises the possibility of such studies being performed on individual patient samples prepared by needle biopsy.     

Effect of essential fatty acids on circulating T cell subsets in patients with colorectal cancer

The effect of essential fatty acids (EFA), given orally as dietary supplements, on the responsiveness in vitro of peripheral blood lymphocytes (PBL), to the mitogen concanavalin A have been studied in 10 patients with localized and 14 patients with advanced colorectal cancer. The degree of lymphocyte activation was assessed by measuring the amount of tritiated [³H]thymidine incorporated into newly synthesised lymphocyte DNA. The results were expressed as stimulation indices. T cell responses to concanavalin A stimulation showed a significant reduction of stimulation indices following EFA supplementation, in both the localized (P=0.026) and advanced (P=0.016) tumour groups, when compared with pretreatment activity in vitro. Mixing experiments, using EFA-supplemented and non-EFA-supplemented lymphocytes with concanavalin A, suggest no enhancement of T suppressor cell activity. Cell surface marker analysis (fluorescence-activated cell sorting for CD phenotyping) revealed a reduction of absolute numbers of CD4⁺ and CD8⁺ lymphocytes following EFA supplementation. The stimulation indices returned to presupplementation values 3 months following cessation of EFA intake. There was no significant change of these indices in the control (no EFA supplementation) advanced tumour group tested. This study suggests that EFA supplementation in patients with colorectal cancer selectively reduces circulating PBL. and T cell subset (including suppressor cells) numbers and/or activity. Such effects may have an important outcome in patients with malignant disease.This work was supported by grants from the Grampian Health Broad, the Royal College of Surgeons of Edinburgh, and Scottish Hospital Endownment Research Trust     

25(OH)D3 and 1,25(OH)2D3 serum concentration and breast tissue expression of 1alpha-hydroxylase, 24-hydroxylase and Vitamin D receptor in women with and without breast cancer

1,25(OH)2D3 is an antiproliferative agent that may inhibit proliferation of breast cancer (BC) cells in vitro and BC development in animals. Epidemiological studies have shown a high incidence of BC in people less exposed to solar rays. To unravel the role of Vitamin D3 in BC patients, we have investigated serum levels of 25(OH)D3 and its active form 1,25(OH)2D3 as well as tissue expression of 1alpha-hydroxylase, 24-hydroxylase, and Vitamin D-receptor (VDR), determined by semiquantitative RT-PCR, in 88 Brazilian BC patients and 35 women without cancer (submitted to mammoplasties or resection of benign lesions). Median age of women with and without cancer was 51 and 46 years, respectively, and the majority of BC patients were classified as clinical stage II (67%). Although no differences in 25(OH)D3 serum concentration were found, 1,25(OH)2D3 (40+/-21 pg/ml) levels in BC patients were lower than in women without cancer (53+/-23). Our results indicate that 24-hydroxylase, VDR and 1alpha-hydroxylase mRNA tissue expression is similar in both groups and no correlation between 24-hydroxylase, 1alpha-hydroxylase, and VDR expression in breast tumors was found. A low 1,25(OH)2D3 serum concentration seems to be associated to breast cancer, however, the mechanism involved in this regulation is still unclear.     

Development of a method for assessing micronucleus induction in a 3D human skin model (EpiDerm)

To meet the requirements of the EU 7th Amendment to the Cosmetics Directive, manufacturers of cosmetics products will need to ascertain the safety of ingredients using non-animal methods. Starting in 2009, in vivo genotoxicity tests for cosmetics ingredients will not be allowed. Skin is a target area of interest for many cosmetic products because of its relatively high exposure. Therefore, it would be beneficial to have a non-animal, skin-based genotoxicity assay, especially one that utilized human skin in vitro. In this paper, we describe the development of a reproducible micronucleus assay that uses EpiDerm engineered human skin constructs (MatTek Corp., Ashland, MA). We describe methods for isolating single cells from the 3D skin model and for processing the cells for microscopic analysis of micronuclei (MN). In addition, since little was known about the kinetics of the dividing keratinocytes in the EpiDerm model, we evaluated whether cytochalasin B (Cyt-B) could be used to distinguish the population of dividing cells allowing the development of a micronucleus assay in binucleated cells. We found that the frequency of binucleated cells increased both with time and with increasing concentration of Cyt-B. After a 48-h exposure, 30-50% binucleated cells were reproducibly obtained. Finally, we evaluated micronucleus induction using the model genotoxicants mitomycin C (MMC) and vinblastine sulfate (VB). The background frequency of MN is very low and reproducible in this model, and statistically significant increases in the frequency of micronucleated cells were induced by both MMC and VB. These are initial steps in developing a routine "in vivo-like" assay for chromosomal damage in human tissue. It is hoped that other investigators utilize these methods to further the understanding of this potentially valuable new non-animal method.