A Novel 3-D Mineralized Tumor Model to Study Breast Cancer Bone Metastasis

Metastatic bone disease is a frequent cause of morbidity in patients with advanced breast cancer, but the role of the bone mineral hydroxyapatite (HA) in this process remains unclear. We have developed a novel mineralized 3-D tumor model and have employed this culture system to systematically investigate the pro-metastatic role of HA under physiologically relevant conditions in vitro.MDA-MB231 breast cancer cells were cultured within non-mineralized or mineralized polymeric scaffolds fabricated by a gas foaming-particulate leaching technique. Tumor cell adhesion, proliferation, and secretion of pro-osteoclastic interleukin-8 (IL-8) was increased in mineralized tumor models as compared to non-mineralized tumor models, and IL-8 secretion was more pronounced for bone-specific MDA-MB231 subpopulations relative to lung-specific breast cancer cells. These differences were pathologically significant as conditioned media collected from mineralized tumor models promoted osteoclastogenesis in an IL-8 dependent manner. Finally, drug testing and signaling studies with transforming growth factor beta (TGFbeta) confirmed the clinical relevance of our culture system and revealed that breast cancer cell behavior is broadly affected by HA.Our results indicate that HA promotes features associated with the neoplastic and metastatic growth of breast carcinoma cells in bone and that IL-8 may play an important role in this process. The developed mineralized tumor models may help to reveal the underlying cellular and molecular mechanisms that may ultimately enable more efficacious therapy of patients with advanced breast cancer

The Emergence of Virtual Tumor Boards in Neuro-Oncology: Opportunities and Challenges.

Background Virtual tumor board (VTB) platforms are an important aspect of cancer management. They enable easier access to a multidisciplinary team of experts. To deliver high-quality cancer care, it is necessary to coordinate numerous therapies and providers, share technical knowledge, and maintain open lines of communication among all professionals involved. The VTB is an essential tool in the diagnosis and treatment of brain cancer. For patients with glioma and brain metastases, multidisciplinary tumor board guidelines should guide diagnosis and therapy throughout the course of the illness. VTBs are an emerging resource across various cancer care networks in the United States. Methodology We performed a systematic search of all VTBs incorporating a platform designed for this specific role. We reviewed the records of the Genomet VTB, the Medical University of South Carolina (MUSC) VTB, and Xcures VTB. Summary data examined included the year of launch, demographics, characteristics of cases, average response time, advantages, and how they handle protected health information. Results Overall, 30% of VTBs examined were launched in 2017. All had a Health Insurance Portability and Accountability Act-compliant online environment. On a review of Xcures records, the median age of the female patients was 57 years and the median age of the male patients was 55 years. The data showed that 44% (4.4 out of every 10 patients) with a confirmed treatment chose the VTB integrated option. Overall, 76% of patients in the Xcures registry had primary central nervous system tumors, with at least 556 patients in the tumor registry which included 46% glioblastoma cases (96% primary, 4% secondary). In the MUSC VTB project, 112 thoracic tumor cases and nine neuro-oncology cases were reviewed. The tumor board met weekly, and the average response time was within 24 hours of case review and presentation. The Genomet VTB de-identifies all patient information; this is a virtual platform primarily focused on neuro-oncology cases. Cases involved a median of five specialists most commonly neuro-oncologists, neurosurgeons, radiation oncologists, molecular pathologists, and neuroradiologists. The case review revealed an age range of six months to 84 years (mean age = 44.5 years), with 69.6% males and 30.4% females, 43.5% glioblastomas, 8.7% adenocarcinomas, and 8.7% infratentorial tumors. The average response time observed in all cases was ≤24 hours. Conclusions VTBs allow for quicker expert analysis of cases. This has resulted in an accelerated number of cases reviewed with a shortened communication time. More studies are needed to gain additional insights into user engagement metrics

Clinical relevance of 3-D mineralized tumor models.

<p>(A) 1833 bone-metastatic MDA-MB231 cells secrete more IL-8 than parental and 4175 lung-metastatic cells in 2-D culture. (B) Mineralized scaffold culture increases IL-8 secretion for all MDA-MB231 populations, but this response is significantly increased in bone-metastatic cells as compared to parental and lung-metastatic cells (**p<0.01, p<0.05). (C) TGFβ1 up-regulates IL-8 secretion in non-mineralized, but not mineralized 3-D cultures of MDA-MB231. (D) In contrast, TGFβ1 increases IL-11 secretion more significantly in mineralized than non-mineralized cultures (p<0.05). (E) Tumor cells cultured within mineralized scaffolds exhibit increased growth suppression in response to ibandronate both when directly exposed to the drug in the dosed interval (days 1–3) and after the drug was removed during the undosed interval (days 3–5). (F) Live/dead staining with calcein (green) and propidium iodide (red) confirmed increased cell death in response to ibandronate in mineralized cultures relative to non-mineralized cultures. Scale bars represent 100 µm.</p

Physicochemical characterization of scaffolds.

<p>(A) HA in biomineralized scaffolds is available for cellular interactions as EDS analysis indicates Ca and P at the porous surface of the scaffolds, while no mineral was detected for non-mineralized control scaffolds. (B) MicroCT scans indicate that HA is uniformly distributed throughout biomineralized scaffolds, but was not present non-mineralized control scaffolds. (C) Incorporation of HA did not alter the scaffold microarchitecture relative to non-mineralized control scaffolds as indicated by visualization via brightfield microscopy. Scale bars represent 2 mm. (D) Image analysis of high-resolution brightfield microscopy images indicates that pore size and polymer wall thickness are similar for both biomineralized and non-mineralized scaffolds.</p

Osteoclastogenesis in response to HA-dependent IL-8 signaling.

<p>(A) Tumor cells cultured within mineralized scaffolds up-regulated secretion of IL-8 relative to culture within non-mineralized control scaffolds, while no effect was detected for VEGF and IL-11 secretion (*p<0.05). Error bars for IL-11 are large due to low IL-11 secretion. (B) Blockade of IL-8 signaling by addition of a neutralizing antibody inhibited the pro-osteoclastic effect of conditioned media collected from mineralized tumors (MIN+) to levels comparable to non-mineralized cultures (MIN-) (*p<0.05). (C) Transwell assays with conditioned media indicate that tumor cells cultured within mineralized scaffolds (MIN+) increase the motility of RAW 264.7 relative to all other conditions. Inhibition with a function blocking antibody suggested that this effect was IL-8 dependent. (D) Colorimetric analysis of Ca-release indicates that IL-8 neutralization in media collected from mineralized tumor models (Ab/MIN+) results in a much more pronounced decrease in osteoclast activity as compared to media collected from non-mineralized scaffold cultures (Ab/MIN-). (E) Micrographs of osteoclast-mediated pit formation on bone mineral surface in the presence of conditioned media from mineralized tumor models with and without IL-8 antibody. Scale bars represent 200 µm. (F) Quantification of pit formation in response to the different media in the presence and absence of functional IL-8 signaling.</p

Effect of HA on breast cancer cell adhesion.

<p>(A) Analysis of von Kossa stained histological cross-sections indicates that MDA-MB231 cells (stained red) penetrate into the center of mineralized scaffolds (HA stained black), while this is not the case for non-mineralized control scaffolds. Arrows and asterisks indicate representative scaffold walls and pores, respectively. Scale bars represent 200 µm. (B) MDA-MB231 breast cancer cells adhere more efficiently to mineralized scaffolds than non-mineralized scaffolds (**p<0.01), and pre-incubation of MDA-MB231 cells with RGD peptide inhibits enhanced adhesion to mineralized scaffolds (**p<0.01). Error bars are small where not visible. (C) Fibronectin adsorption within the polymer scaffold is increased in mineralized scaffolds (lane 2) relative to non-mineralized scaffolds (lane 1) as indicated by Western Blot analysis of scaffold lysates.</p

Osteoclastogenesis in response to conditioned media.

<p>(A) Conditioned media collected from mineralized tumor models increased RAW 264.7 osteoclastogenesis relative to conditioned media collected from non-mineralized tumor models as revealed by TRAP staining of large multinucleated cells. (B) Quantification of TRAP+ cells indicated that culture media collected from mineralized scaffold cultures (MIN+) promoted RAW 264.7 osteoclastogenesis relative to control media (cDMEM) and media collected from non-mineralized scaffold cultures (MIN-) in a manner that was similar to osteoclastic RANKL. Asterisks [*p<0.05, **p<0.01] and pound signs [#p<0.05, ##p<0.01] indicate statistical significance with respect to ‘cDMEM’ and ‘MIN-’, respectively. (C) Conditioned media collected from mineralized models (MIN+) enhances the resorptive activity of RAW 264.7 relative to all other tested conditions as indicated by 2-D culture on calcium phosphate disks and subsequent analysis of calcium release by a colorimetric assay.</p

Stromal EGF and igf-I together modulate plasticity of disseminated triple-negative breast tumors

The causes for malignant progression of disseminated tumors and the reasons recurrence rates differ in women with different breast cancer subtypes are unknown. Here, we report novel mechanisms of tumor plasticity that are mandated by microenvironmental factors and show that recurrence rates are not strictly due to cell-intrinsic properties. Specifically, outgrowth of the same population of incipient tumors is accelerated in mice with triple-negative breast cancer (TNBC) relative to those with luminal breast cancer. Systemic signals provided by overt TNBCs cause the formation of a tumor-supportive microenvironment enriched for EGF and insulin-like growth factor-I (IGF-I) at distant indolent tumor sites. Bioavailability of EGF and IGF-I enhances the expression of transcription factors associated with pluripotency, proliferation, and epithelial-mesenchymal transition. Combinatorial therapy with EGF receptor and IGF-I receptor inhibitors prevents malignant progression. These results suggest that plasticity and recurrence rates can be dictated by host systemic factors and offer novel therapeutic potential for patients with TNBC