Neutrophils resist ferroptosis and promote breast cancer metastasis through aconitate decarboxylase 1.

Metastasis causes breast cancer-related mortality. Tumor-infiltrating neutrophils (TINs) inflict immunosuppression and promote metastasis. Therapeutic debilitation of TINs may enhance immunotherapy, yet it remains a challenge to identify therapeutic targets highly expressed and functionally essential in TINs but under-expressed in extra-tumoral neutrophils. Here, using single-cell RNA sequencing to compare TINs and circulating neutrophils in murine mammary tumor models, we identified aconitate decarboxylase 1 (Acod1) as the most upregulated metabolic enzyme in mouse TINs and validated high Acod1 expression in human TINs. Activated through the GM-CSF-JAK/STAT5-C/EBPß pathway, Acod1 produces itaconate, which mediates Nrf2-dependent defense against ferroptosis and upholds the persistence of TINs. Acod1 ablation abates TIN infiltration, constrains metastasis (but not primary tumors), bolsters antitumor T cell immunity, and boosts the efficacy of immune checkpoint blockade. Our findings reveal how TINs escape from ferroptosis through the Acod1-dependent immunometabolism switch and establish Acod1 as a target to offset immunosuppression and improve immunotherapy against metastasis.

Host obesity alters the ovarian tumor immune microenvironment and impacts response to standard of care chemotherapy.

BACKGROUND: The majority of women with epithelial ovarian cancer (OvCa) are diagnosed with metastatic disease, resulting in a poor 5-year survival of 31%. Obesity is a recognized non-infectious pandemic that increases OvCa incidence, enhances metastatic success and reduces survival. We have previously demonstrated a link between obesity and OvCa metastatic success in a diet-induced obesity mouse model wherein a significantly enhanced tumor burden was associated with a decreased M1/M2 tumor-associated macrophage ratio (Liu Y et al. Can, Res. 2015; 75:5046-57). METHODS: The objective of this study was to use pre-clinical murine models of diet-induced obesity to evaluate the effect of a high fat diet (HFD) on response to standard of care chemotherapy and to assess obesity-associated changes in the tumor microenvironment. Archived tumor tissues from ovarian cancer patients of defined body mass index (BMI) were also evaluated using multiplexed immunofluorescence analysis of immune markers. RESULTS: We observed a significantly diminished response to standard of care paclitaxel/carboplatin chemotherapy in HFD mice relative to low fat diet (LFD) controls. A corresponding decrease in the M1/M2 macrophage ratio and enhanced tumor fibrosis were observed both in murine DIO studies and in human tumors from women with BMI > 30. CONCLUSIONS: Our data suggest that the reported negative impact of obesity on OvCa patient survival may be due in part to the effect of the altered M1/M2 tumor-associated macrophage ratio and enhanced fibrosis on chemosensitivity. These data demonstrate a contribution of host obesity to ovarian tumor progression and therapeutic response and support future combination strategies targeting macrophage polarization and/or fibrosis in the obese host.

Advanced Glycation End Products as a Potential Target for Restructuring the Ovarian Cancer Microenvironment: A Pilot Study.

Ovarian cancer is the sixth leading cause of cancer-related death in women, and both occurrence and mortality are increased in women over the age of 60. There are documented age-related changes in the ovarian cancer microenvironment that have been shown to create a permissive metastatic niche, including the formation of advanced glycation end products, or AGEs, that form crosslinks between collagen molecules. Small molecules that disrupt AGEs, known as AGE breakers, have been examined in other diseases, but their efficacy in ovarian cancer has not been evaluated. The goal of this pilot study is to target age-related changes in the tumor microenvironment with the long-term aim of improving response to therapy in older patients. Here, we show that AGE breakers have the potential to change the omental collagen structure and modulate the peritoneal immune landscape, suggesting a potential use for AGE breakers in the treatment of ovarian cancer.

Degradomic Identification of Membrane Type 1-Matrix Metalloproteinase as an ADAMTS9 and ADAMTS20 Substrate.

The secreted metalloproteases ADAMTS9 and ADAMTS20 are implicated in extracellular matrix proteolysis and primary cilium biogenesis. Here, we show that clonal gene-edited RPE-1 cells in which ADAMTS9 was inactivated, and which constitutively lack ADAMTS20 expression, have morphologic characteristics distinct from parental RPE-1 cells. To investigate underlying proteolytic mechanisms, a quantitative terminomics method, terminal amine isotopic labeling of substrates was used to compare the parental and gene-edited RPE-1 cells and their medium to identify ADAMTS9 substrates. Among differentially abundant neo-amino (N) terminal peptides arising from secreted and transmembrane proteins, a peptide with lower abundance in the medium of gene-edited cells suggested cleavage at the Tyr314-Gly315 bond in the ectodomain of the transmembrane metalloprotease membrane type 1-matrix metalloproteinase (MT1-MMP), whose mRNA was also reduced in gene-edited cells. This cleavage, occurring in the MT1-MMP hinge, that is, between the catalytic and hemopexin domains, was orthogonally validated both by lack of an MT1-MMP catalytic domain fragment in the medium of gene-edited cells and restoration of its release from the cell surface by reexpression of ADAMTS9 and ADAMTS20 and was dependent on hinge O-glycosylation. A C-terminally semitryptic MT1-MMP peptide with greater abundance in WT RPE-1 medium identified a second ADAMTS9 cleavage site in the MT1-MMP hemopexin domain. Consistent with greater retention of MT1-MMP on the surface of gene-edited cells, pro-MMP2 activation, which requires cell surface MT1-MMP, was increased. MT1-MMP knockdown in gene-edited ADAMTS9/20-deficient cells restored focal adhesions but not ciliogenesis. The findings expand the web of interacting proteases at the cell surface, suggest a role for ADAMTS9 and ADAMTS20 in regulating cell surface activity of MT1-MMP, and indicate that MT1-MMP shedding does not underlie their observed requirement in ciliogenesis.

Maternal obesity driven changes in collagen linearity of breast extracellular matrix induces invasive mammary epithelial cell phenotype.

Obesity has been linked with numerous health issues as well as an increased risk of breast cancer. Although effects of direct obesity in patient outcomes is widely studied, effects of exposure to obesity-related systemic influences in utero have been overlooked. In this study, we investigated the effect of multigenerational obesity on epithelial cell migration and invasion using decellularized breast tissues explanted from normal female mouse pups from a diet induced multigenerational obesity mouse model. We first studied the effect of multigenerational diet on the mechanical properties, adipocyte size, and collagen structure of these mouse breast tissues, and then, examined the migration and invasion behavior of normal (KTB-21) and cancerous (MDA-MB-231) human mammary epithelial cells on the decellularized matrices from each diet group. Breast tissues of mice whose dams had been fed with high-fat diet exhibited larger adipocytes and thicker and curvier collagen fibers, but only slightly elevated elastic modulus and inflammatory cytokine levels. MDA-MB-231 cancer cell motility and invasion were significantly greater on the decellularized matrices from mice whose dams were fed with high-fat diet. A similar trend was observed with normal KTB-21 cells. Our results showed that the collagen curvature was the dominating factor on this enhanced motility and stretching the matrices to equalize the collagen fiber linearity of the matrices ameliorated the observed increase in cell migration and invasion in the mice that were exposed to a high-fat diet in utero. Previous studies indicated an increase in serum leptin concentration for those children born to an obese mother. We generated extracellular matrices using primary fibroblasts exposed to various concentrations of leptin. This produced curvier ECM and increased breast cancer cell motility for cells seeded on the decellularized ECM generated with increasing leptin concentration. Our study shows that exposure to obesity in utero is influential in determining the extracellular matrix structure, and that the resultant change in collagen curvature is a critical factor in regulating the migration and invasion of breast cancer cells.

Another Wrinkle with Age: Aged Collagen and Intra-peritoneal Metastasis of Ovarian Cancer.

Background: Age is the most significant risk factor for ovarian cancer (OvCa), the deadliest gynecologic malignancy. Metastasizing OvCa cells adhere to the omentum, a peritoneal structure rich in collagen, adipocytes, and immune cells. Ultrastructural changes in the omentum and the omental collagen matrix with aging have not been evaluated. Aim: The aim of this study was to test the hypothesis that age-related changes in collagen in the ovarian tumor microenvironment promote OvCa metastatic success in the aged host. Methods/Results: Young (3-6 months) and aged mice (20-23 months) were used to study the role of aging in metastatic success. Intra-peritoneal (IP) injection of ID8Trp53 -/- ovarian cancer cells showed enhanced IP dissemination in aged vs young mice. In vitro assays using purified collagen demonstrated reduced collagenolysis of aged fibers, as visualized using scanning electron microscopy (SEM) and quantified with a hydroxyproline release assay. Omental tumors in young and aged mice showed similar collagen deposition; however enhanced intra-tumoral collagen remodeling was seen in aged mice probed with a biotinylated collagen hybridizing peptide (CHP). In contrast, second harmonic generation (SHG) microscopy showed significant differences in collagen fiber structure and organization in omental tissue and SEM demonstrated enhanced omental fenestration in aged omenta. Combined SHG and Alexa Fluor-CHP microscopy in vivo demonstrated that peri-tumoral collagen was remodeled more extensively in young mice. This collagen population represents truly aged host collagen, in contrast to intra-tumoral collagen that is newly synthesized, likely by cancer associated fibroblasts (CAFs). Conclusions: Our results demonstrate that tumors in an aged host can grow with minimal collagen remodeling, while tumors in the young host must remodel peri-tumoral collagen to enable effective proliferation, providing a mechanism whereby age-induced ultrastructural changes in collagen and collagen-rich omenta establish a permissive pre-metastatic niche contributing to enhanced OvCa metastatic success in the aged host.

Biocompatible, Multi-Mode, Fluorescent, T2 MRI Contrast Magnetoelectric-Silica Nanoparticles (MagSiNs), for On-Demand Doxorubicin Delivery to Metastatic Cancer Cells.

There is a need to improve current cancer treatment regimens to reduce systemic toxicity, to positively impact the quality-of-life post-treatment. We hypothesized the negation of off-target toxicity of anthracyclines (e.g., Doxorubicin) by delivering Doxorubicin on magneto-electric silica nanoparticles (Dox-MagSiNs) to cancer cells. Dox-MagSiNs were completely biocompatible with all cell types and are therapeutically inert till the release of Doxorubicin from the MagSiNs at the cancer cells location. The MagSiNs themselves are comprised of biocompatible components with a magnetostrictive cobalt ferrite core (4−6 nm) surrounded by a piezoelectric fused silica shell of 1.5 nm to 2 nm thickness. The MagSiNs possess T2-MRI contrast properties on par with RESOVIST™ due to their cobalt ferrite core. Additionally, the silica shell surrounding the core was volume loaded with green or red fluorophores to fluorescently track the MagSiNs in vitro. This makes the MagSiNs a suitable candidate for trackable, drug nanocarriers. We used metastatic triple-negative breast cancer cells (MDAMB231), ovarian cancer cells (A2780), and prostate cancer cells (PC3) as our model cancer cell lines. Human umbilical vein endothelial cells (HUVEC) were used as control cell lines to represent blood-vessel cells that suffer from the systemic toxicity of Doxorubicin. In the presence of an external magnetic field that is 300× times lower than an MRI field, we successfully nanoporated the cancer cells, then triggered the release of 500 nM of doxorubicin from Dox-MagSiNs to successfully kill >50% PC3, >50% A2780 cells, and killed 125% more MDAMB231 cells than free Dox.HCl. In control HUVECs, the Dox-MagSiNs did not nanoporate into the HUVECS and did not exhibited any cytotoxicity at all when there was no triggered release of Dox.HCl. Currently, the major advantages of our approach are, (i) the MagSiNs are biocompatible in vitro and in vivo; (ii) the label-free nanoporation of Dox-MagSiNs into cancer cells and not the model blood vessel cell line; (iii) the complete cancellation of the cytotoxicity of Doxorubicin in the Dox-MagSiNs form; (iv) the clinical impact of such a nanocarrier will be that it will be possible to increase the current upper limit for cumulative-dosages of anthracyclines through multiple dosing, which in turn will improve the anti-cancer efficacy of anthracyclines.

Development and evaluation of ActSeq: A targeted next-generation sequencing panel for clinical oncology use.

PURPOSE: The demand for high-throughput genetic profiling of somatic mutations in cancer tissues is growing. We sought to establish a targeted next generation sequencing (NGS) panel test for clinical oncology practice. METHODS: Customized probes were designed to capture exonic regions of 141 genes selected for the panel, which was aimed for the detection of clinically actionable genetic variations in cancer, including KRAS, NRAS, BRAF, ALK, ROS1, KIT and EGFR. The size of entire targeted regions is 0.8 Mb. Library preparation used NEBNext Ultra II FS kit coupled with target enrichment. Paired-end sequencing was run on Illumina NextSeq 500 at a read length of 150 nt. A bioinformatics workflow focusing on single nucleotide variant and short insertions and deletions (SNV/indel) discovery was established using open source, in-house and commercial software tools. Standard reference DNA samples were used in testing the sensitivity and precision and limit of detection in variant calling. RESULTS: The general performance of the panel was observed in pilot runs. Average total reads per sample ranged from 30 million to 48 million, 73% ~82% unique reads. All runs had more than 99% average mapping rate. Mean target coverage ranged from 727x to 879x. Depth of coverage at 50x or more reached 87% of targeted region and 60% of targeted region received 500x or more coverage depth. Using OncoSpan HD827 DNA, which bears 144 variants (SNV/indel) from 80 genes that are within the targeted region on the panel, our somatic variant calling pipeline reached 97% sensitivity and 100% precision respectively, with near 48 million reads. High concordance with orthogonal approaches in variant detection was further verified with 7 cancer cell lines and 45 clinical specimens. CONCLUSION: We developed a NGS panel with a focus on clinically actionable gene mutations and validated the performance in library construction, sequencing and variant calling. High concordance with reference materials and orthogonal mutation detection was observed.

Host Mesothelin Expression Increases Ovarian Cancer Metastasis in the Peritoneal Microenvironment.

Mesothelin (MSLN), a glycoprotein normally expressed by mesothelial cells, is overexpressed in ovarian cancer (OvCa) suggesting a role in tumor progression, although the biological function is not fully understood. OvCa has a high mortality rate due to diagnosis at advanced stage disease with intraperitoneal metastasis. Tumor cells detach from the primary tumor as single cells or multicellular aggregates (MCAs) and attach to the mesothelium of organs within the peritoneal cavity producing widely disseminated secondary lesions. To investigate the role of host MSLN in the peritoneal cavity we used a mouse model with a null mutation in the MSLN gene (MSLNKO). The deletion of host MSLN expression modified the peritoneal ultrastructure resulting in abnormal mesothelial cell surface architecture and altered omental collagen fibril organization. Co-culture of murine OvCa cells with primary mesothelial cells regardless of MSLN expression formed compact MCAs. However, co-culture with MSLNKO mesothelial cells resulted in smaller MCAs. An allograft tumor study, using wild-type mice (MSLNWT) or MSLNKO mice injected intraperitoneally with murine OvCa cells demonstrated a significant decrease in peritoneal metastatic tumor burden in MSLNKO mice compared to MSLNWT mice. Together, these data support a role for host MSLN in the progression of OvCa metastasis.

Aged Breast Extracellular Matrix Drives Mammary Epithelial Cells to an Invasive and Cancer-Like Phenotype.

Age is a major risk factor for cancer. While the importance of age related genetic alterations in cells on cancer progression is well documented, the effect of aging extracellular matrix (ECM) has been overlooked. This study shows that the aging breast ECM alone is sufficient to drive normal human mammary epithelial cells (KTB21) to a more invasive and cancer-like phenotype, while promoting motility and invasiveness in MDA-MB-231 cells. Decellularized breast matrix from aged mice leads to loss of E-cadherin membrane localization in KTB21 cells, increased cell motility and invasion, and increased production of inflammatory cytokines and cancer-related proteins. The aged matrix upregulates cancer-related genes in KTB21 cells and enriches a cell subpopulation highly expressing epithelial-mesenchymal transition-related genes. Lysyl oxidase knockdown reverts the aged matrix-induced changes to the young levels; it relocalizes E-cadherin to cell membrane, and reduces cell motility, invasion, and cytokine production. These results show for the first time that the aging ECM harbors key biochemical, physical, and mechanical cues contributing to invasive and cancer-like behavior in healthy and cancer mammary cells. Differential response of cells to young and aged ECMs can lead to identification of new targets for cancer treatment and prevention.

Metalloproteinases in Ovarian Cancer.

Proteases play a crucial role in the progression and metastasis of ovarian cancer. Pericellular protein degradation and fragmentation along with remodeling of the extracellular matrix (ECM) is accomplished by numerous proteases that are present in the ovarian tumor microenvironment. Several proteolytic processes have been linked to cancer progression, particularly those facilitated by the matrix metalloproteinase (MMP) family. These proteases have been linked to enhanced migratory ability, extracellular matrix breakdown, and development of support systems for tumors. Several studies have reported the direct involvement of MMPs with ovarian cancer, as well as their mechanisms of action in the tumor microenvironment. MMPs play a key role in upregulating transcription factors, as well as the breakdown of structural proteins like collagen. Proteolytic mechanisms have been shown to enhance the ability of ovarian cancer cells to migrate and adhere to secondary sites allowing for efficient metastasis. Furthermore, angiogenesis for tumor growth and development of metastatic implants is influenced by upregulation of certain proteases, including MMPs. While proteases are produced normally in vivo, they can be upregulated by cancer-associated mutations, tumor-microenvironment interaction, stress-induced catecholamine production, and age-related pathologies. This review outlines the important role of proteases throughout ovarian cancer progression and metastasis.

In vivo selection of highly metastatic human ovarian cancer sublines reveals role for AMIGO2 in intra-peritoneal metastatic regulation.

The majority of women with ovarian cancer are diagnosed with metastatic disease, therefore elucidating molecular events that contribute to successful metastatic dissemination may identify additional targets for therapeutic intervention and thereby positively impact survival. Using two human high grade serous ovarian cancer cell lines with inactive TP53 and multiple rounds of serial in vivo passaging, we generated sublines with significantly accelerated intra-peritoneal (IP) growth. Comparative analysis of the parental and IP sublines identified a common panel of differentially expressed genes. The most highly differentially expressed gene, upregulated by 60-65-fold in IP-selected sublines, was the type I transmembrane protein AMIGO2. As the role of AMIGO2 in ovarian cancer metastasis remains unexplored, CRISPR/Cas9 was used to reduce AMIGO2 expression, followed by in vitro and in vivo functional analyses. Knockdown of AMIGO2 modified the sphere-forming potential of ovarian cancer cells, reduced adhesion and invasion in vitro, and significantly attenuated IP metastasis. These data highlight AMIGO2 as a new target for a novel anti-metastatic therapeutic approach aimed at blocking cohesion, survival, and adhesion of metastatic tumorspheres.

Unexpected PD-L1 immune evasion mechanism in TNBC, ovarian, and other solid tumors by DR5 agonist antibodies.

Lack of effective immune infiltration represents a significant barrier to immunotherapy in solid tumors. Thus, solid tumor-enriched death receptor-5 (DR5) activating antibodies, which generates tumor debulking by extrinsic apoptotic cytotoxicity, remains a crucial alternate therapeutic strategy. Over past few decades, many DR5 antibodies moved to clinical trials after successfully controlling tumors in immunodeficient tumor xenografts. However, DR5 antibodies failed to significantly improve survival in phase-II trials, leading in efforts to generate second generation of DR5 agonists to supersize apoptotic cytotoxicity in tumors. Here we have discovered that clinical DR5 antibodies activate an unexpected immunosuppressive PD-L1 stabilization pathway, which potentially had contributed to their limited success in clinics. The DR5 agonist stimulated caspase-8 signaling not only activates ROCK1 but also undermines proteasome function, both of which contributes to increased PD-L1 stability on tumor cell surface. Targeting DR5-ROCK1-PD-L1 axis markedly increases immune effector T-cell function, promotes tumor regression, and improves overall survival in animal models. These insights have identified a potential clinically viable combinatorial strategy to revive solid cancer immunotherapy using death receptor agonism.

Lipid Regulatory Proteins as Potential Therapeutic Targets for Ovarian Cancer in Obese Women.

Obesity has become a recognized global epidemic that is associated with numerous comorbidities including type II diabetes, cardiovascular disease, hypertension, and cancer incidence and progression. Ovarian cancer (OvCa) has a unique mechanism of intra-peritoneal metastasis, already present in 80% of women at the time of diagnosis, making it the fifth leading cause of death from gynecological malignancy. Meta-analyses showed that obesity increases the risk of OvCa progression, leads to enhanced overall and organ-specific tumor burden, and adversely effects survival of women with OvCa. Recent data discovered that tumors grown in mice fed on a western diet (40% fat) have elevated lipid levels and a highly increased expression level of sterol regulatory element binding protein 1 (SREBP1). SREBP1 is a master transcription factor that regulates de novo lipogenesis and lipid homeostasis, and induces lipogenic reprogramming of tumor cells. Elevated SREBP1 levels are linked to cancer cell proliferation and metastasis. This review will summarize recent findings to provide a current understanding of lipid regulatory proteins in the ovarian tumor microenvironment with emphasis on SREBP1 expression in the obese host, the role of SREBP1 in cancer progression and metastasis, and potential therapeutic targeting of SREBPs and SREBP-pathway genes in treating cancers, particularly in the context of host obesity.

Lysophosphatidic acid modulates ovarian cancer multicellular aggregate assembly and metastatic dissemination.

Epithelial ovarian cancer (EOC) metastasis occurs by exfoliation of cells and multicellular aggregates (MCAs) from the tumor into the peritoneal cavity, adhesion to and retraction of peritoneal mesothelial cells and subsequent anchoring. Elevated levels of lysophosphatidic acid (LPA) have been linked to aberrant cell proliferation, oncogenesis, and metastasis. LPA disrupts junctional integrity and epithelial cohesion in vitro however, the fate of free-floating cells/MCAs and the response of host peritoneal tissues to LPA remain unclear. EOC MCAs displayed significant LPA-induced changes in surface ultrastructure with the loss of cell surface protrusions and poor aggregation, resulting in increased dissemination of small clusters compared to untreated control MCAs. LPA also diminished the adhesive capacity of EOC single cells and MCAs to murine peritoneal explants and impaired MCA survival and mesothelial clearance competence. Peritoneal tissues from healthy mice injected with LPA exhibited enhanced mesothelial surface microvilli. Ultrastructural alterations were associated with restricted peritoneal susceptibility to metastatic colonization by single cells as well as epithelial-type MCAs. The functional consequence is an LPA-induced dissemination of small mesenchymal-type clusters, promoting a miliary mode of peritoneal seeding that complicates surgical removal and is associated with worse prognosis.

Ascites-induced compression alters the peritoneal microenvironment and promotes metastatic success in ovarian cancer.

The majority of women with recurrent ovarian cancer (OvCa) develop malignant ascites with volumes that can reach > 2 L. The resulting elevation in intraperitoneal pressure (IPP), from normal values of 5 mmHg to as high as 22 mmHg, causes striking changes in the loading environment in the peritoneal cavity. The effect of ascites-induced changes in IPP on OvCa progression is largely unknown. Herein we model the functional consequences of ascites-induced compression on ovarian tumor cells and components of the peritoneal microenvironment using a panel of in vitro, ex vivo and in vivo assays. Results show that OvCa cell adhesion to the peritoneum was increased under compression. Moreover, compressive loads stimulated remodeling of peritoneal mesothelial cell surface ultrastructure via induction of tunneling nanotubes (TNT). TNT-mediated interaction between peritoneal mesothelial cells and OvCa cells was enhanced under compression and was accompanied by transport of mitochondria from mesothelial cells to OvCa cells. Additionally, peritoneal collagen fibers adopted a more linear anisotropic alignment under compression, a collagen signature commonly correlated with enhanced invasion in solid tumors. Collectively, these findings elucidate a new role for ascites-induced compression in promoting metastatic OvCa progression.

Large-Scale Image Analysis for Investigating Spatio-Temporal Changes in Nuclear DNA Damage Caused by Nitrogen Atmospheric Pressure Plasma Jets.

The effective clinical application of atmospheric pressure plasma jet (APPJ) treatments requires a well-founded methodology that can describe the interactions between the plasma jet and a treated sample and the temporal and spatial changes that result from the treatment. In this study, we developed a large-scale image analysis method to identify the cell-cycle stage and quantify damage to nuclear DNA in single cells. The method was then tested and used to examine spatio-temporal distributions of nuclear DNA damage in two cell lines from the same anatomic location, namely the oral cavity, after treatment with a nitrogen APPJ. One cell line was malignant, and the other, nonmalignant. The results showed that DNA damage in cancer cells was maximized at the plasma jet treatment region, where the APPJ directly contacted the sample, and declined radially outward. As incubation continued, DNA damage in cancer cells decreased slightly over the first 4 h before rapidly decreasing by approximately 60% at 8 h post-treatment. In nonmalignant cells, no damage was observed within 1 h after treatment, but damage was detected 2 h after treatment. Notably, the damage was 5-fold less than that detected in irradiated cancer cells. Moreover, examining damage with respect to the cell cycle showed that S phase cells were more susceptible to DNA damage than either G1 or G2 phase cells. The proposed methodology for large-scale image analysis is not limited to APPJ post-treatment applications and can be utilized to evaluate biological samples affected by any type of radiation, and, more so, the cell-cycle classification can be used on any cell type with any nuclear DNA staining.

Host Wnt5a Potentiates Microenvironmental Regulation of Ovarian Cancer Metastasis.

The noncanonical Wnt ligand Wnt5a is found in high concentrations in ascites of women with ovarian cancer. In this study, we elucidated the role of Wnt5a in ovarian cancer metastasis. Wnt5a promoted ovarian tumor cell adhesion to peritoneal mesothelial cells as well as migration and invasion, leading to colonization of peritoneal explants. Host components of the ovarian tumor microenvironment, notably peritoneal mesothelial cells and visceral adipose, secreted Wnt5a. Conditional knockout of host WNT5A significantly reduced peritoneal metastatic tumor burden. Tumors formed in WNT5A knockout mice had elevated cytotoxic T cells, increased M1 macrophages, and decreased M2 macrophages, indicating that host Wnt5a promotes an immunosuppressive microenvironment. The Src family kinase Fgr was identified as a downstream effector of Wnt5a. These results highlight a previously unreported role for host-expressed Wnt5a in ovarian cancer metastasis and suggest Fgr as a novel target for inhibition of ovarian cancer metastatic progression.Significance: This study establishes host-derived Wnt5a, expressed by peritoneal mesothelial cells and adipocytes, as a primary regulator of ovarian cancer intraperitoneal metastatic dissemination and identifies Fgr kinase as novel target for inhibition of metastasis.

The Tumor Microenvironment of High Grade Serous Ovarian Cancer.

The Special Issue on high grade serous ovarian cancer (HGSOC) and the contribution of the tumor micro-environment (TME) consisted of reviews contributed by leaders in the ovarian cancer (OC) field. [...].

Modeling the effect of ascites-induced compression on ovarian cancer multicellular aggregates.

Epithelial ovarian cancer (EOC) is the most lethal gynecological malignancy. EOC dissemination is predominantly via direct extension of cells and multicellular aggregates (MCAs) into the peritoneal cavity, which adhere to and induce retraction of peritoneal mesothelium and proliferate in the submesothelial matrix to generate metastatic lesions. Metastasis is facilitated by the accumulation of malignant ascites (500 ml to >2 l), resulting in physical discomfort and abdominal distension, and leading to poor prognosis. Although intraperitoneal fluid pressure is normally subatmospheric, an average intraperitoneal pressure of 30 cmH2O (22.1 mmHg) has been reported in women with EOC. In this study, to enable experimental evaluation of the impact of high intraperitoneal pressure on EOC progression, two new in vitro model systems were developed. Initial experiments evaluated EOC MCAs in pressure vessels connected to an Instron to apply short-term compressive force. A Flexcell Compression Plus system was then used to enable longer-term compression of MCAs in custom-designed hydrogel carriers. Results show changes in the expression of genes related to epithelial-mesenchymal transition as well as altered dispersal of compressed MCAs on collagen gels. These new model systems have utility for future analyses of compression-induced mechanotransduction and the resulting impact on cellular responses related to intraperitoneal metastatic dissemination.This article has an associated First Person interview with the first authors of the paper.