Controlled mechanical loading affects the osteocyte transcriptome in porcine trabecular bone in situ.

INTRODUCTION: Osteocytes modulate bone adaptation in response to mechanical stimuli imparted by the deforming bone tissue in which they are encased by communicating with osteoclasts and osteoblasts as well as other osteocytes in the lacuna-canalicular network through secreted cytokines and chemokines. Understanding the transcriptional response of osteocytes to mechanical stimulation in situ could identify new targets to inhibit bone loss or enhance bone formation in the presence of diseases like osteoporosis or metastatic cancer. We compared the mechanically regulated transcriptional response of osteocytes in trabecular bone following one or three days of controlled mechanical loading. METHODS: Porcine trabecular bone explants were cultured in a bioreactor for 48 h and subsequently loaded twice a day for one day or 3 days. RNA was isolated and sequenced, and the Tuxedo suite was used to identify differentially expressed genes and pathway analysis was conducted using Ingenuity Pathway Analysis (IPA). RESULTS: There were about 4000 differentially expressed genes following in situ culture relative to fresh bone. One hundred six genes were differentially expressed between the loaded and non-loaded groups following one day of loading compared to 913 genes after 3 d of loading. Only 45 of these were coincident between the two time points, indicating an evolving transcriptome. Clustering and principal component analysis indicated differences between the loaded and non-loaded groups after 3 d of loading. DISCUSSION: With sustained loading, there was a nine-fold increase in the number of differentially expressed genes, suggesting that osteocytes respond to loading through sequential activation of downstream genes in the same pathways. The differentially expressed genes were related to osteoarthritis, osteocyte, and chondrocyte signaling pathways. We noted that NFkB and TNF signaling are affected by early loading and this may drive downstream effects on the mechanobiological response. Moreover, these genes may regulate catabolic effects of mechanical disuse through their actions on pre-osteoclasts in the bone marrow niche.

Disease-driven engineering of peptide-targeted DM1 loaded liposomal nanoparticles for enhanced efficacy in treating multiple myeloma by exploring DM1 prodrug chemistry.

Here, we report a CD138 receptor targeting liposomal formulation (TNP[Prodrug-4]) that achieved efficacious tumor growth inhibition in treating multiple myeloma by overcoming the dose limiting severe toxicity issues of a highly potent drug, Mertansine (DM1). Despite the promising potential to treat various cancers, due to poor solubility and pharmacokinetic profile, DM1's translation to the clinic has been unsatisfactory. We hypothesized that the optimal prodrug chemistry would promote efficient loading of the prodrug into targeted nanoparticles and achieve controlled release following endocytosis by the cancer cells, consequently, accomplish the most potent tumor growth inhibition. We evaluated four functional linker chemistries for synthesizing DM1-Prodrug molecules and evaluated their stability and cancer cell toxicity in vitro. It was determined that the phosphodiester moiety, as part of nanoparticle formulations, demonstrated most favorable characteristics with an IC50 of ∼16 nM. Nanoparticle formulations of Prodrug-4 enabled its administration at 8-fold higher dosage of equivalent free drug while remaining below maximum tolerated dose. Importantly, TNP[Prodrug-4] achieved near complete inhibition of tumor growth (∼99% by day 10) compared to control, without displaying noticeable systemic toxicity. TNP[Prodrug-4] promises a formulation that could potentially make DM1 treatment available for wider clinical applications with a long-term goal for better patient outcomes.

Aquaporins: New players in breast cancer progression and treatment response.

Aquaporins (AQPs) are a family of small transmembrane proteins that selectively transport water and other small molecules and ions following an osmotic gradient across cell plasma membranes. This enables them to regulate numerous functions including water homeostasis, fat metabolism, proliferation, migration, and adhesion. Previous structural and functional studies highlight a strong biological relationship between AQP protein expression, localization, and key biological functions in normal and cancer tissues, where aberrant AQP expression correlates with tumorigenesis and metastasis. In this review, we discuss the roles of AQP1, AQP3, AQP4, AQP5, and AQP7 in breast cancer progression and metastasis, including the role of AQPs in the tumor microenvironment, to highlight potential contributions of stromal-derived to epithelial-derived AQPs to breast cancer. Emerging evidence identifies AQPs as predictors of response to cancer therapy and as targets for increasing their sensitivity to treatment. However, these studies have not evaluated the requirements for protein structure on AQP function within the context of breast cancer. We also examine how AQPs contribute to a patient's response to cancer treatment, existing AQP inhibitors and how AQPs could serve as novel predictive biomarkers of therapy response in breast cancer. Future studies also should evaluate AQP redundancy and compensation as mechanisms used to overcome aberrant AQP function. This review highlights the need for additional research into how AQPs contribute molecularly to therapeutic resistance and by altering the tumor microenvironment.

Quality control recommendations for RNASeq using FFPE samples based on pre-sequencing lab metrics and post-sequencing bioinformatics metrics.

BACKGROUND: Formalin-fixed, paraffin-embedded (FFPE) tissues have many advantages for identification of risk biomarkers, including wide availability and potential for extended follow-up endpoints. However, RNA derived from archival FFPE samples has limited quality. Here we identified parameters that determine which FFPE samples have the potential for successful RNA extraction, library preparation, and generation of usable RNAseq data. METHODS: We optimized library preparation protocols designed for use with FFPE samples using seven FFPE and Fresh Frozen replicate pairs, and tested optimized protocols using a study set of 130 FFPE biopsies from women with benign breast disease. Metrics from RNA extraction and preparation procedures were collected and compared with bioinformatics sequencing summary statistics. Finally, a decision tree model was built to learn the relationship between pre-sequencing lab metrics and qc pass/fail status as determined by bioinformatics metrics. RESULTS: Samples that failed bioinformatics qc tended to have low median sample-wise correlation within the cohort (Spearman correlation < 0.75), low number of reads mapped to gene regions (< 25 million), or low number of detectable genes (11,400 # of detected genes with TPM > 4). The median RNA concentration and pre-capture library Qubit values for qc failed samples were 18.9 ng/ul and 2.08 ng/ul respectively, which were significantly lower than those of qc pass samples (40.8 ng/ul and 5.82 ng/ul). We built a decision tree model based on input RNA concentration, input library qubit values, and achieved an F score of 0.848 in predicting QC status (pass/fail) of FFPE samples. CONCLUSIONS: We provide a bioinformatics quality control recommendation for FFPE samples from breast tissue by evaluating bioinformatic and sample metrics. Our results suggest a minimum concentration of 25 ng/ul FFPE-extracted RNA for library preparation and 1.7 ng/ul pre-capture library output to achieve adequate RNA-seq data for downstream bioinformatics analysis.

The effect of marrow secretome and culture environment on the rate of metastatic breast cancer cell migration in two and three dimensions.

Metastasis is responsible for over 90% of cancer-related deaths, and bone is the most common site for breast cancer metastasis. Metastatic breast cancer cells home to trabecular bone, which contains hematopoietic and stromal lineage cells in the marrow. As such, it is crucial to understand whether bone or marrow cells enhance breast cancer cell migration toward the tissue. To this end, we quantified the migration of MDA-MB-231 cells toward human bone in two- and three-dimensional (3D) environments. First, we found that the cancer cells cultured on tissue culture plastic migrated toward intact trabecular bone explants at a higher rate than toward marrow-deficient bone or devitalized bone. Leptin was more abundant in conditioned media from the cocultures with intact explants, while higher levels of IL-1ß, IL-6, and TNFα were detected in cultures with both intact bone and cancer cells. We further verified that the cancer cells migrated into bone marrow using a bioreactor culture system. Finally, we studied migration toward bone in 3D gelatin. Migration speed did not depend on stiffness of this homogeneous gel, but many more dendritic-shaped cancer cells oriented and migrated toward bone in stiffer gels than softer gels, suggesting a coupling between matrix mechanics and chemotactic signals.

In vivo evaluation of CD38 and CD138 as targets for nanoparticle-based drug delivery in multiple myeloma.

BACKGROUND: Drug-loaded nanoparticles have established their benefits in the fight against multiple myeloma; however, ligand-targeted nanomedicine has yet to successfully translate to the clinic due to insufficient efficacies reported in preclinical studies. METHODS: In this study, liposomal nanoparticles targeting multiple myeloma via CD38 or CD138 receptors are prepared from pre-synthesized, purified constituents to ensure increased consistency over standard synthetic methods. These nanoparticles are then tested both in vitro for uptake to cancer cells and in vivo for accumulation at the tumor site and uptake to tumor cells. Finally, drug-loaded nanoparticles are tested for long-term efficacy in a month-long in vivo study by tracking tumor size and mouse health. RESULTS: The targeted nanoparticles are first optimized in vitro and show increased uptake and cytotoxicity over nontargeted nanoparticles, with CD138-targeting showing superior enhancement over CD38-targeted nanoparticles. However, biodistribution and tumor suppression studies established CD38-targeted nanoparticles to have significantly increased in vivo tumor accumulation, tumor cell uptake, and tumor suppression over both nontargeted and CD138-targeted nanoparticles due to the latter's poor selectivity. CONCLUSION: These results both highlight a promising cancer treatment option in CD38-targeted nanoparticles and emphasize that targeting success in vitro does not necessarily translate to success in vivo.

Stiff stroma increases breast cancer risk by inducing the oncogene ZNF217.

Women with dense breasts have an increased lifetime risk of malignancy that has been attributed to a higher epithelial density. Quantitative proteomics, collagen analysis, and mechanical measurements in normal tissue revealed that stroma in the high-density breast contains more oriented, fibrillar collagen that is stiffer and correlates with higher epithelial cell density. microRNA (miR) profiling of breast tissue identified miR-203 as a matrix stiffness-repressed transcript that is downregulated by collagen density and reduced in the breast epithelium of women with high mammographic density. Culture studies demonstrated that ZNF217 mediates a matrix stiffness- and collagen density-induced increase in Akt activity and mammary epithelial cell proliferation. Manipulation of the epithelium in a mouse model of mammographic density supported a causal relationship between stromal stiffness, reduced miR-203, higher levels of the murine homolog Zfp217, and increased Akt activity and mammary epithelial proliferation. ZNF217 was also increased in the normal breast epithelium of women with high mammographic density, correlated positively with epithelial proliferation and density, and inversely with miR-203. The findings identify ZNF217 as a potential target toward which preexisting therapies, such as the Akt inhibitor triciribine, could be used as a chemopreventive agent to reduce cancer risk in women with high mammographic density.

Aquaporin-7 Regulates the Response to Cellular Stress in Breast Cancer.

The complex yet interrelated connections between cancer metabolism, gene expression, and oncogenic driver genes have the potential to identify novel biomarkers and drug targets with prognostic and therapeutic value. Here we effectively integrated metabolomics and gene expression data from breast cancer mouse models through a novel unbiased correlation-based network analysis. This approach identified 35 metabolite and 34 gene hubs with the most network correlations. These hubs have prognostic value and are likely integral to tumor metabolism and breast cancer. The gene hub Aquaporin-7 (Aqp7), a water and glycerol channel, was identified as a novel regulator of breast cancer. AQP7 was prognostic of overall survival in patients with breast cancer. In mouse breast cancer models, reduced expression of Aqp7 caused reduced primary tumor burden and lung metastasis. Metabolomics and complex lipid profiling of cells and tumors with reduced Aqp7 revealed significantly altered lipid metabolism, glutathione metabolism, and urea/arginine metabolism compared with controls. These data identify AQP7 as a critical regulator of metabolic and signaling responses to environmental cellular stresses in breast cancer, highlighting AQP7 as a potential cancer-specific therapeutic vulnerability. SIGNIFICANCE: Aquaporin-7 is identified as a critical regulator of nutrient availability and signaling that responds to cellular stresses, making it an attractive therapeutic target in breast cancer. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/19/4071/F1.large.jpg.

Engineering peptide-targeted liposomal nanoparticles optimized for improved selectivity for HER2-positive breast cancer cells to achieve enhanced in vivo efficacy.

Here, we report rationally engineered peptide-targeted liposomal doxorubicin nanoparticles that have an enhanced selectivity for HER2-positive breast tumor cells with high purity, reproducibility, and precision in controlling stoichiometry of targeting peptides. To increase HER2-positive tumor cell selective drug delivery, we optimized the two most important design parameters, peptide density and linker length, via systematic evaluations of their effects on both in vitro cellular uptake and in vivo tumor accumulation and cellular uptake. The optimally designed nanoparticles were finally evaluated for their tumor inhibition efficacy using in vivo MMTV-neu transplantation mouse model. In vitro, we demonstrated that ~1% peptide density and EG8 linker were optimal parameters for targeted nanoparticle formulations to enhance HER2-positive cancer cellular uptake while preventing non-selectivity. In vivo results demonstrated that at 0.5% peptide density, enhancement of tumor cell uptake over non-targeted nanoparticles was ~2.7 fold and ~3.4 fold higher for targeted nanoparticles with EG8 and EG18 linker, respectively, while their accumulation levels at tumor tissue were similar to the non-targeted nanoparticles. These results were consistent with in vivo efficacy outcomes that ~90% tumor growth inhibition was achieved by Dox-loaded HER2 receptor targeted nanoparticles, TNPHER2pep, over control while all nanoparticle formulations minimized overall systemic toxicity relative to free Dox. This study highlights the significance of understanding and optimizing the effects of liposomal nanoparticle design parameters for enhancement of tumor selectivity to achieve improved in vivo therapeutic outcomes.

A Statistical Approach of Background Removal and Spectrum Identification for SERS Data.

SERS (surface-enhanced Raman scattering) enhances the Raman signals, but the plasmonic effects are sensitive to the chemical environment and the coupling between nanoparticles, resulting in large and variable backgrounds, which make signal matching and analyte identification highly challenging. Removing background is essential, but existing methods either cannot fit the strong fluctuation of the SERS spectrum or do not consider the spectra's shape change across time. Here we present a new statistical approach named SABARSI that overcomes these difficulties by combining information from multiple spectra. Further, after efficiently removing the background, we have developed the first automatic method, as a part of SABARSI, for detecting signals of molecules and matching signals corresponding to identical molecules. The superior efficiency and reproducibility of SABARSI are shown on two types of experimental datasets.

Untargeted Tumor Metabolomics with Liquid Chromatography-Surface-Enhanced Raman Spectroscopy.

Metabolomics is a powerful systems biology approach that monitors changes in biomolecule concentrations to diagnose and monitor health and disease. However, leading metabolomics technologies, such as NMR and mass spectrometry (MS), access only a small portion of the metabolome. Now an approach is presented that uses the high sensitivity and chemical specificity of surface-enhanced Raman scattering (SERS) for online detection of metabolites from tumor lysates following liquid chromatography (LC). The results demonstrate that this LC-SERS approach has metabolite detection capabilities comparable to the state-of-art LC-MS but suggest a selectivity for the detection of a different subset of metabolites. Analysis of replicate LC-SERS experiments exhibit reproducible metabolite patterns that can be converted into barcodes, which can differentiate different tumor models. Our work demonstrates the potential of LC-SERS technology for metabolomics-based diagnosis and treatment of cancer.

The CXCL5/CXCR2 axis is sufficient to promote breast cancer colonization during bone metastasis.

Bone is one of the most common sites for metastasis across cancers. Cancer cells that travel through the vasculature and invade new tissues can remain in a non-proliferative dormant state for years before colonizing the metastatic site. Switching from dormancy to colonization is the rate-limiting step of bone metastasis. Here we develop an ex vivo co-culture method to grow cancer cells in mouse bones to assess cancer cell proliferation using healthy or cancer-primed bones. Profiling soluble factors from conditioned media identifies the chemokine CXCL5 as a candidate to induce metastatic colonization. Additional studies using CXCL5 recombinant protein suggest that CXCL5 is sufficient to promote breast cancer cell proliferation and colonization in bone, while inhibition of its receptor CXCR2 with an antagonist blocks proliferation of metastatic cancer cells. This study suggests that CXCL5 and CXCR2 inhibitors may have efficacy in treating metastatic bone tumors dependent on the CXCL5/CXCR2 axis.

Death effector domain-containing protein induces vulnerability to cell cycle inhibition in triple-negative breast cancer.

Lacking targetable molecular drivers, triple-negative breast cancer (TNBC) is the most clinically challenging subtype of breast cancer. In this study, we reveal that Death Effector Domain-containing DNA-binding protein (DEDD), which is overexpressed in > 60% of TNBCs, drives a mitogen-independent G1/S cell cycle transition through cytoplasm localization. The gain of cytosolic DEDD enhances cyclin D1 expression by interacting with heat shock 71 kDa protein 8 (HSC70). Concurrently, DEDD interacts with Rb family proteins and promotes their proteasome-mediated degradation. DEDD overexpression renders TNBCs vulnerable to cell cycle inhibition. Patients with TNBC have been excluded from CDK 4/6 inhibitor clinical trials due to the perceived high frequency of Rb-loss in TNBCs. Interestingly, our study demonstrated that, irrespective of Rb status, TNBCs with DEDD overexpression exhibit a DEDD-dependent vulnerability to combinatorial treatment with CDK4/6 inhibitor and EGFR inhibitor in vitro and in vivo. Thus, our study provided a rationale for the clinical application of CDK4/6 inhibitor combinatorial regimens for patients with TNBC.

Potassium channel activity controls breast cancer metastasis by affecting ß-catenin signaling.

Potassium ion channels are critical in the regulation of cell motility. The acquisition of cell motility is an essential parameter of cancer metastasis. However, the role of K+ channels in cancer metastasis has been poorly studied. High expression of the hG1 gene, which encodes for Kv11.1 channel associates with good prognosis in estrogen receptor-negative breast cancer (BC). We evaluated the efficacy of the Kv11.1 activator NS1643 in arresting metastasis in a triple negative breast cancer (TNBC) mouse model. NS1643 significantly reduces the metastatic spread of breast tumors in vivo by inhibiting cell motility, reprogramming epithelial-mesenchymal transition via attenuation of Wnt/ß-catenin signaling and suppressing cancer cell stemness. Our findings provide important information regarding the clinical relevance of potassium ion channel expression in breast tumors and the mechanisms by which potassium channel activity can modulate tumor biology. Findings suggest that Kv11.1 activators may represent a novel therapeutic approach for the treatment of metastatic estrogen receptor-negative BC. Ion channels are critical factor for cell motility but little is known about their role in metastasis. Stimulation of the Kv11.1 channel suppress the metastatic phenotype in TNBC. This work could represent a paradigm-shifting approach to reducing mortality by targeting a pathway that is central to the development of metastases.

Metabolomics of oncogene-specific metabolic reprogramming during breast cancer.

BACKGROUND: The complex yet interrelated connections between cancer metabolism and oncogenic driver genes are relatively unexplored but have the potential to identify novel biomarkers and drug targets with prognostic and therapeutic value. The goal of this study was to identify global metabolic profiles of breast tumors isolated from multiple transgenic mouse models and to identify unique metabolic signatures driven by these oncogenes. METHODS: Using mass spectrometry (GC-MS, LC-MS/MS, and capillary zone electrophoresis (CZE)-MS platforms), we quantified and compared the levels of 374 metabolites in breast tissue from normal and transgenic mouse breast cancer models overexpressing a panel of oncogenes (PyMT, PyMT-DB, Wnt1, Neu, and C3-TAg). We also compared the mouse metabolomics data to published human metabolomics data already linked to clinical data. RESULTS: Through analysis of our metabolomics data, we identified metabolic differences between normal and tumor breast tissues as well as metabolic differences unique to each initiating oncogene. We also quantified the metabolic profiles of the mammary fat pad versus mammary epithelium by CZE-MS/MS. However, the differences between the tissues did not account for the majority of the metabolic differences between the normal mammary gland and breast tumor tissues. Therefore, the differences between the cohorts were unlikely due to cellular heterogeneity. Of the mouse models used in this study, C3-TAg was the only cohort with a tumor metabolic signature composed of ten metabolites that had significant prognostic value in breast cancer patients. Gene expression analysis identified candidate genes that may contribute to the metabolic reprogramming. CONCLUSIONS: This study identifies oncogene-induced metabolic reprogramming within mouse breast tumors and compares the results to that of human breast tumors, providing a unique look at the relationship between and clinical value of oncogene initiation and metabolism during breast cancer.

Discoidin domain receptor 1 (DDR1) ablation promotes tissue fibrosis and hypoxia to induce aggressive basal-like breast cancers.

The discoidin domain receptor 1 (DDR1) is overexpressed in breast carcinoma cells. Low DDR1 expression is associated with worse relapse-free survival, reflecting its controversial role in cancer progression. We detected DDR1 on luminal cells but not on myoepithelial cells of DDR1+/+ mice. We found that DDR1 loss compromises cell adhesion, consistent with data that older DDR1-/- mammary glands had more basal/myoepithelial cells. Basal cells isolated from older mice exerted higher traction forces than the luminal cells, in agreement with increased mammary branches observed in older DDR1-/- mice and higher branching by their isolated organoids. When we crossed DDR1-/- mice with MMTV-PyMT mice, the PyMT/DDR1-/- mammary tumors grew faster and had increased epithelial tension and matricellular fibrosis with a more basal phenotype and increased lung metastases. DDR1 deletion induced basal differentiation of CD90+CD24+ cancer cells, and the increase in basal cells correlated with tumor cell mitoses. K14+ basal cells, including K8+K14+ cells, were increased adjacent to necrotic fields. These data suggest that the absence of DDR1 provides a growth and adhesion advantage that favors the expansion of basal cells, potentiates fibrosis, and enhances necrosis/hypoxia and basal differentiation of transformed cells to increase their aggression and metastatic potential.

Tumor infiltrating leukocyte density is independent of tumor grade and molecular subtype in aggressive breast cancer of Western Kenya.

BACKGROUND: Tumors commonly are infiltrated by leukocytes, or tumor infiltrating leukocytes (TILs). It remains unclear, however, if the density and type of individual TILs has a direct or simply correlative role in promoting poor prognosis in breast cancer patients. Breast cancer in Kenyan women is aggressive with presentation at a young age, with advanced grade (grade III), large tumor size (>2.0 cm), and poor prognosis. We previously observed that the tumors were predominantly estrogen receptor positive (ER+) but also included both a high percentage of triple negative tumors and also increased immune cell infiltration within the tumors. We used breast tumor tissues from each patient to make tissue microarrays that were then stained for leukocyte and myeloid markers including CD4, CD8, CD20, CD25, CD68, and CD163 using immunohistochemical techniques. The immune cell infiltration into the cancer tissue included increased numbers of macrophages (CD68+), helper T cells (CD4+), and CD25+ lymphocytes compared to benign tissue. RESULTS: This study characterized the grade, molecular subtypes, and proliferation index of these tumors and determined if TIL density was enriched across any of these factors. We analyzed 49 malignant patient tissue samples for this study. The patient population had a mean age of 51.9 years. The tumors analyzed were heterogeneous by grade: grade I (6%), grade II (47%), and grade III (39%). Most patients presented with large tumors (>2.0 cm) (69%). We classified the tumors into molecular subtypes based on clinical marker expression. Based on this analysis, the molecular subtype distribution was heterogeneous with luminal B (41%), basal/triple negative (TN) (37%), luminal A (14%) and HER2 (8%) breast cancer subtypes. While the basal/TN subtype had a much higher proliferative index (Ki-67+) than did the other molecular subtypes, we did not see a significant correlation between TIL density and either subtype or tumor grade. Therefore, TIL density is independent of molecular subtype and grade. CONCLUSION: This study identified a Kenyan patient cohort that develops large, high-grade tumors primarily of the luminal B and basal molecular subtypes. After analyzing the TILs within these tumors, we found that immune cell infiltration of these tumors correlated with increased proliferation but not grade or molecular subtype. Future research is required to determine if the aberrant recruitment of TILs to tumors contributes to cancer progression and response to cancer treatments.

The AKT inhibitor triciribine in combination with paclitaxel has order-specific efficacy against Zfp217-induced breast cancer chemoresistance.

We previously identified the transcription factor ZNF217 (human) / Zfp217 (mouse) as an oncogene and prognostic indicator of reduced survival, increased metastasis, and reduced response to therapy in breast cancer patients. Here we investigated the role of Zfp217 in chemotherapy resistance. Preclinical animal models of Zfp217 overexpression were treated with a combination therapy of the microtubule inhibitor epothilone B, doxorubicin (Adriamycin), and cyclophosphamide (EAC). Tumors overexpressing Zfp217 increased their tumor burden compared to control tumors after treatment and accumulated a mammary gland progenitor cell population (K8+K14+). To overcome this chemoresistance after ZNF217 overexpression, we treated tumors ± Zfp217 overexpression with paclitaxel and triciribine, a nucleoside analog and AKT inhibitor that kills cells that overexpress ZNF217. Treatment order critically impacted the efficacy of the therapy. Combination treatment of triciribine followed by paclitaxel (TCN→PAC) inhibited tumor burden and increased survival in tumors that overexpressed Zfp217, whereas single agent or combination treatment in the reverse order (PAC→TCN) did not improve response. Analysis of these tumors and patient-derived tumor xenograft tumors treated with the same therapies suggested that Zfp217 overexpression in tumors contributes both to decreased microvessel density and vessel maturity, while TCN→PAC tumors overexpressing Zfp217 showed improved vessel maturity.

Bone: A Fertile Soil for Cancer Metastasis.

Bone is one of the most common and most dangerous sites for metastatic growth across cancer types, and bone metastasis remains incurable. Unfortunately, the processes by which cancers preferentially metastasize to bone are still not well understood. In this review, we summarize the morphological features, physical properties, and cell signaling events that make bone a unique site for metastasis and bone remodeling. The signaling crosstalk between the tumor cells and bone cells begins a vicious cycle - a self-sustaining feedback loop between the tumor cells and the bone microenvironment composed of osteoclasts, osteoblasts, other bone marrow cells, bone matrix, and vasculature to support both tumor growth and bone destruction. Through this crosstalk, bone provides a fertile microenvironment that can harbor dormant tumor cells, sometimes for long periods, and support their growth by releasing cytokines as the bone matrix is destroyed, similar to providing nutrients for a seed to germinate in soil. However, few models exist to study the late stages of bone colonization by metastatic tumor cells. We describe some of the current methodologies used to study bone metastasis, highlighting the limitations of these methods and alternative future strategies to be used to study bone metastasis. While <i>in vivo</i> animal and patient studies may provide the gold standard for studying metastasis, <i>ex vivo</i> models can be used as an alternative to enable more controlled experiments designed to study the late stages of bone metastasis.

Aggressive breast cancer in western Kenya has early onset, high proliferation, and immune cell infiltration.

BACKGROUND: Breast cancer incidence and mortality vary significantly among different nations and racial groups. African nations have the highest breast cancer mortality rates in the world, even though the incidence rates are below those of many nations. Differences in disease progression suggest that aggressive breast tumors may harbor a unique molecular signature to promote disease progression. However, few studies have investigated the pathology and clinical markers expressed in breast tissue from regional African patient populations. METHODS: We collected 68 malignant and 89 non-cancerous samples from Kenyan breast tissue. To characterize the tumors from these patients, we constructed tissue microarrays (TMAs) from these tissues. Sections from these TMAs were stained and analyzed using immunohistochemistry to detect clinical breast cancer markers, including estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor 2 receptor (HER2) status, Ki67, and immune cell markers. RESULTS: Thirty-three percent of the tumors were triple negative (ER-, PR-, HER2-), 59% were ER+, and almost all tumors analyzed were HER2-. Seven percent of the breast cancer patients were male, and 30% were <40 years old at diagnosis. Cancer tissue had increased immune cell infiltration with recruitment of CD163+ (M2 macrophage), CD25+ (regulatory T lymphocyte), and CD4+ (T helper) cells compared to non-cancer tissue. CONCLUSIONS: We identified clinical biomarkers that may assist in identifying therapy strategies for breast cancer patients in western Kenya. Estrogen receptor status in particular should lead initial treatment strategies in these breast cancer patients. Increased CD25 expression suggests a need for additional treatment strategies designed to overcome immune suppression by CD25+ cells in order to promote the antitumor activity of CD8+ cytotoxic T cells.