Genetic and Functional Modifications Associated with Ovarian Cancer Cell Aggregation and Limited Culture Conditions.

The aggregation of cancer cells provides a survival signal for disseminating cancer cells; however, the underlying molecular mechanisms have yet to be elucidated. Using qPCR gene arrays, this study investigated the changes in cancer-specific genes as well as genes regulating mitochondrial quality control, metabolism, and oxidative stress in response to aggregation and hypoxia in our progressive ovarian cancer models representing slow- and fast-developing ovarian cancer. Aggregation increased the expression of anti-apoptotic, stemness, epithelial-mesenchymal transition (EMT), angiogenic, mitophagic, and reactive oxygen species (ROS) scavenging genes and functions, and decreased proliferation, apoptosis, metabolism, and mitochondrial content genes and functions. The incorporation of stromal vascular cells (SVF) from obese mice into the spheroids increased DNA repair and telomere regulatory genes that may represent a link between obesity and ovarian cancer risk. While glucose had no effect, glutamine was essential for aggregation and supported proliferation of the spheroid. In contrast, low glucose and hypoxic culture conditions delayed adhesion and outgrowth capacity of the spheroids independent of their phenotype, decreased mitochondrial mass and polarity, and induced a shift of mitochondrial dynamics towards mitophagy. However, these conditions did not reduce the appearance of polarized mitochondria at adhesion sites, suggesting that adhesion signals that either reversed mitochondrial fragmentation or induced mitobiogenesis can override the impact of low glucose and oxygen levels. Thus, the plasticity of the spheroids' phenotype supports viability during dissemination, allows for the adaptation to changing conditions such as oxygen and nutrient availability. This may be critical for the development of an aggressive cancer phenotype and, therefore, could represent druggable targets for clinical interventions.

Adaptation of metabolism to multicellular aggregation, hypoxia and obese stromal cell incorporation as potential measure of survival of ovarian metastases.

Ovarian metastases exfoliate from the primary tumor and it is thought that aggregation supports their survival in the peritoneal cavity during dissemination but the underlying mechanisms are not clearly identified. We have previously shown that ovarian cancer cells acquire an increasingly glycolytic and metabolic flexible phenotype during progression. In the present study, we investigated how hypoxia, aggregation, and the incorporation of the obese stromal vascular fraction (SVF) affect cellular metabolism and the response to common anti-cancer and anti-diabetic drugs. Our results show a reduction of glucose uptake, lactate secretion, cellular respiration and ATP synthesis in response to hypoxia and aggregation, suggesting that the observed reduced proliferation of cells aggregated into spheroids is the result of a down-regulation of respiration. Recruitment of SVF to spheroids increased the spheroids invasive capacity but reduced respiration only in the most aggressive cells. Further, aggregation and hypoxia reduced the response to the metabolic drugs AICAR and metformin, and the chemotherapeutic agents cisplatin and paclitaxel. Our results suggest that the adaptation of cellular metabolism may contribute to enhanced survival under non-permissive conditions, and that these metabolic alterations may provide targets for future interventions that aim to enhance the survival of women with metastatic ovarian cancer.

A novel ultralow conductivity electromanipulation buffer improves cell viability and enhances dielectrophoretic consistency.

Cell separation has become a critical diagnostic, research, and treatment tool for personalized medicine. Despite significant advances in cell separation, most widely used applications require the use of multiple, expensive antibodies to known markers in order to identify subpopulations of cells for separation. Dielectrophoresis (DEP) provides a biophysical separation technique that can target cell subpopulations based on phenotype without labels and return native cells for downstream analysis. One challenge in employing any DEP device is the sample being separated must be transferred into an ultralow conductivity medium, which can be detrimental in retaining cells' native phenotypes for separation. Here, we measured properties of traditional DEP reagents and determined that after just 1-2 h of exposure and subsequent culture, cells' viability was significantly reduced below 50%. We developed and tested a novel buffer (Cyto Buffer) that achieved 6 weeks of stable shelf-life and demonstrated significantly improved viability and physiological properties. We then determined the impact of Cyto Buffer on cells' dielectric properties and morphology and found that cells retained properties more similar to that of their native media. Finally, we vetted Cyto Buffer's usability on a cell separation platform (Cyto R1) to determine combined efficacy for cell separations. Here, more than 80% of cells from different cell lines were recovered and were determined to be >70% viable following exposure to Cyto Buffer, flow stimulation, electromanipulation, and downstream collection and growth. The developed buffer demonstrated improved opportunities for electrical cell manipulation, enrichment, and recovery for next generation cell separations.

Self-aligned microfluidic contactless dielectrophoresis device fabricated by single-layer imprinting on cyclic olefin copolymer.

The trapping and deflection of biological cells by dielectrophoresis (DEP) at field non-uniformities in a microfluidic device is often conducted in a contactless dielectrophoresis (cDEP) mode, wherein the electrode channel is in a different layer than the sample channel, so that field penetration through the interceding barrier causes DEP above critical cut-off frequencies. In this manner, through physical separation of the electrode and sample channels, it is possible to spatially modulate electric fields with no electrode-induced damage to biological cells in the sample channel. However, since this device requires interlayer alignment of the electrode to sample channel and needs to maintain a thin interceding barrier (~ 15 µm) over the entire length over which DEP is needed (~ 1 cm), variations in alignment and microstructure fidelity cause wide variations in cDEP trapping level and frequency response across devices. We present a strategy to eliminate interlayer alignment by fabricating self-aligned electrode and sample channels, simultaneously with the interceding barrier layer (14-µm width and 50-µm depth), using a single-layer imprint and bond process on cyclic olefin copolymer. Specifically, by designing support structures, we preserve fidelity of the high aspect ratio insulating posts in the sample channel and the interceding barrier between the sample and electrode channels over the entire device footprint (~ 1 cm). The device operation is validated based on impedance measurements to quantify field penetration through the interceding barrier and by DEP trapping measurements. The presented fabrication strategy can eventually improve cDEP device manufacturing protocols to enable more reproducible DEP performance. Graphical abstract.

A feasibility study for enrichment of highly aggressive cancer subpopulations by their biophysical properties via dielectrophoresis enhanced with synergistic fluid flow.

A common problem with cancer treatment is the development of treatment resistance and tumor recurrence that result from treatments that kill most tumor cells yet leave behind aggressive cells to repopulate. Presented here is a microfluidic device that can be used to isolate tumor subpopulations to optimize treatment selection. Dielectrophoresis (DEP) is a phenomenon where particles are polarized by an electric field and move along the electric field gradient. Different cell subpopulations have different DEP responses depending on their bioelectrical phenotype, which, we hypothesize, correlate with aggressiveness. We have designed a microfluidic device in which a region containing posts locally distorts the electric field created by an AC voltage and forces cells toward the posts through DEP. This force is balanced with a simultaneous drag force from fluid motion that pulls cells away from the posts. We have shown that by adjusting the drag force, cells with aggressive phenotypes are influenced more by the DEP force and trap on posts while others flow through the chip unaffected. Utilizing single-cell trapping via cell-sized posts coupled with a drag-DEP force balance, we show that separation of similar cell subpopulations may be achieved, a result that was previously impossible with DEP alone. Separated subpopulations maintain high viability downstream, and remain in a native state, without fluorescent labeling. These cells can then be cultured to help select a therapy that kills aggressive subpopulations equally or better than the bulk of the tumor, mitigating resistance and recurrence.

Alternating electric fields (TTFields) in combination with paclitaxel are therapeutically effective against ovarian cancer cells in vitro and in vivo.

Long-term survival rates for advanced ovarian cancer patients have not changed appreciably over the past four decades; therefore, development of new, effective treatment modalities remains a high priority. Tumor Treating Fields (TTFields), a clinically active anticancer modality utilize low-intensity, intermediate frequency, alternating electric fields. The goal of this study was to evaluate the efficacy of combining TTFields with paclitaxel against ovarian cancer cells in vitro and in vivo. In vitro application of TTFields on human ovarian cancer cell lines led to a significant reduction in cell counts as compared to untreated cells. The effect was found to be frequency and intensity dependent. Further reduction in the number of viable cells was achieved when TTFields treatment was combined with paclitaxel. The in vivo effect of the combined treatment was tested in mice orthotopically implanted with MOSE-LTICv cells. In this model, combined treatment led to a significant reduction in tumor luminescence and in tumor weight as compared to untreated mice. The feasibility of effective local delivery of TTFields to the human abdomen was examined using finite element mesh simulations performed using the Sim4life software. These simulations demonstrated that electric fields intensities inside and in the vicinity of the ovaries of a realistic human computational phantom are about 1 and 2 V/cm pk-pk, respectively, which is within the range of intensities required for TTFields effect. These results suggest that prospective clinical investigation of the combination of TTFields and paclitaxel is warranted.

Ovarian tumor-initiating cells display a flexible metabolism.

An altered metabolism during ovarian cancer progression allows for increased macromolecular synthesis and unrestrained growth. However, the metabolic phenotype of cancer stem or tumor-initiating cells, small tumor cell populations that are able to recapitulate the original tumor, has not been well characterized. In the present study, we compared the metabolic phenotype of the stem cell enriched cell variant, MOSE-LFFLv (TIC), derived from mouse ovarian surface epithelial (MOSE) cells, to their parental (MOSE-L) and benign precursor (MOSE-E) cells. TICs exhibit a decrease in glucose and fatty acid oxidation with a concomitant increase in lactate secretion. In contrast to MOSE-L cells, TICs can increase their rate of glycolysis to overcome the inhibition of ATP synthase by oligomycin and can increase their oxygen consumption rate to maintain proton motive force when uncoupled, similar to the benign MOSE-E cells. TICs have an increased survival rate under limiting conditions as well as an increased survival rate when treated with AICAR, but exhibit a higher sensitivity to metformin than MOSE-E and MOSE-L cells. Together, our data show that TICs have a distinct metabolic profile that may render them flexible to adapt to the specific conditions of their microenvironment. By better understanding their metabolic phenotype and external environmental conditions that support their survival, treatment interventions can be designed to extend current therapy regimens to eradicate TICs.

Metabolic changes during ovarian cancer progression as targets for sphingosine treatment.

Tumor cells often exhibit an altered metabolic phenotype. However, it is unclear as to when this switch takes place in ovarian cancer, and the potential for these changes to serve as therapeutic targets in clinical prevention and intervention trials. We used our recently developed and characterized mouse ovarian surface epithelial (MOSE) cancer progression model to study metabolic changes in distinct disease stages. As ovarian cancer progresses, complete oxidation of glucose and fatty acids were significantly decreased, concurrent with increases in lactate excretion and (3)H-deoxyglucose uptake by the late-stage cancer cells, shifting the cells towards a more glycolytic phenotype. These changes were accompanied by decreases in TCA flux but an increase in citrate synthase activity, providing substrates for de novo fatty acid and cholesterol synthesis. Also, uncoupled maximal respiration rates in mitochondria decreased as cancer progressed. Treatment of the MOSE cells with 1.5 µM sphingosine, a bioactive sphingolipid metabolite, decreased citrate synthase activity, increased TCA flux, decreased cholesterol synthesis and glycolysis. Together, our data confirm metabolic changes during ovarian cancer progression, indicate a stage specificity of these changes, and suggest that multiple events in cellular metabolism are targeted by exogenous sphingosine which may be critical for future prevention trials.

Biomechanical profile of cancer stem-like/tumor-initiating cells derived from a progressive ovarian cancer model.

We herein report, for the first time, the mechanical properties of ovarian cancer stem-like/tumor-initiating cells (CSC/TICs). The represented model is a spontaneously transformed murine ovarian surface epithelial (MOSE) cell line that mimics the progression of ovarian cancer from early/non-tumorigenic to late/highly aggressive cancer stages. Elastic modulus measurements via atomic force microscopy (AFM) illustrate that the enriched CSC/TICs population (0.32±0.12kPa) are 46%, 61%, and 72% softer (P<0.0001) than their aggressive late-stage, intermediate, and non-malignant early-stage cancer cells, respectively. Exposure to sphingosine, an anti-cancer agent, induced an increase in the elastic moduli of CSC/TICs by more than 46% (0.47±0.14kPa, P<0.0001). Altogether, our data demonstrate that the elastic modulus profile of CSC/TICs is unique and responsive to anti-cancer treatment strategies that impact the cytoskeleton architecture of cells. These findings increase the chance for obtaining distinctive cell biomechanical profiles with the intent of providing a means for effective cancer detection and treatment control. FROM THE CLINICAL EDITOR: This novel study utilized atomic force microscopy to demonstrate that the elastic modulus profile of cancer stem cell-like tumor initiating cells is unique and responsive to anti-cancer treatment strategies that impact the cytoskeleton of these cells. These findings pave the way to the development of unique means for effective cancer detection and treatment control.

Obesity modulates the cellular and molecular microenvironment in the peritoneal cavity: implication for ovarian cancer risk.

Introduction: Abdominal obesity increases the risk of developing ovarian cancer but the molecular mechanisms of how obesity supports ovarian cancer development remain unknown. Here we investigated the impact of obesity on the immune cell and gene expression profiles of distinct abdominal tissues, focusing on the peritoneal serous fluid (PSF) and the omental fat band (OFB) as critical determinants for the dissemination of ovarian metastases and early metastatic events within the peritoneal cavity. Methods: Female C57BL/6 mice were fed a low-fat (LFD) or a high-fat diet (HFD) for 12 weeks until the body weights in the HFD group were significantly higher and the mice displayed an impaired glucose tolerance. Then the mice were injected with the murine ovarian cancer cells (MOSE-LTICv) while remaining on their diets. After 21 days, the mice were sacrificed, tumor burden was evaluated and tissues were harvested. The immune cell composition of abdominal tissues and changes in gene expression in the PSF and OFB were evaluated by flow cytometry and qPCR RT2-profiler PCR arrays and confirmed by qRT-PCR, respectively. Other peritoneal adipose tissues including parametrial and retroperitoneal white adipose tissues as well as blood were also investigated. Results: While limited effects were observed in the other peritoneal adipose tissues, feeding mice the HFD led to distinct changes in the immune cell composition in the PSF and the OFB: a depletion of B cells but an increase in myeloid-derived suppressor cells (MDSC) and mono/granulocytes, generating pro-inflammatory environments with increased expression of cyto- and chemokines, and genes supporting adhesion, survival, and growth, as well as suppression of apoptosis. This was associated with a higher peritoneal tumor burden compared to mice fed a LFD. Changes in cellular and genetic profiles were often exacerbated by the HFD. There was a large overlap in genes that were modulated by both the HFD and the cancer cells, suggesting that this 'genetic fingerprint' is important for ovarian metastases to the OFB. Discussion: In accordance with the 'seed and soil' theory, our studies show that obesity contributes to the generation of a pro-inflammatory peritoneal environment that supports the survival of disseminating ovarian cancer cells in the PSF and the OFB and enhances the early metastatic adhesion events in the OFB through an increase in extracellular matrix proteins and modulators such as fibronectin 1 and collagen I expression as well as in genes supporting growth and invasion such as Tenacin C. The identified genes could potentially be used as targets for prevention strategies to lower the ovarian cancer risk in women with obesity.

The effects of cancer progression on the viscoelasticity of ovarian cell cytoskeleton structures.

Alterations in the biomechanical properties and cytoskeletal organization of cancer cells in addition to genetic changes have been correlated with their aggressive phenotype. In this study, we investigated changes in the viscoelasticity of mouse ovarian surface epithelial (MOSE) cells, a mouse model for progressive ovarian cancer. We demonstrate that the elasticity of late-stage MOSE cells (0.549 ± 0.281 kPa) were significantly less than that of their early-stage counterparts (1.097 ± 0.632 kPa). Apparent cell viscosity also decreased significantly from early (144.7 ± 102.4 Pa-s) to late stage (50.74 ± 29.72 Pa-s). This indicates that ovarian cells are stiffer and more viscous when they are benign. The increase in cell deformability directly correlates with the progression of a transformed phenotype from a nontumorigenic, benign cell to a tumorigenic, malignant one. The decrease in the level of actin in the cytoskeleton and its organization is directly associated with the changes in cell biomechanical property. FROM THE CLINICAL EDITOR: The authors have investigated changes in the viscoelasticity of mouse ovarian surface epithelial (MOSE) cells and demonstrated that ovarian cells are stiffer and more viscous when they are benign.

Metabolic Reprogramming of Ovarian Cancer Spheroids during Adhesion.

Ovarian cancer remains a deadly disease and its recurrence disease is due in part to the presence of disseminating ovarian cancer aggregates not removed by debulking surgery. During dissemination in a dynamic ascitic environment, the spheroid cells' metabolism is characterized by low respiration and fragmented mitochondria, a metabolic phenotype that may not support secondary outgrowth after adhesion. Here, we investigated how adhesion affects cellular respiration and substrate utilization of spheroids mimicking early stages of secondary metastasis. Using different glucose and oxygen levels, we investigated cellular metabolism at early time points of adherence (24 h and less) comparing slow and fast-developing disease models. We found that adhesion over time showed changes in cellular energy metabolism and substrate utilization, with a switch in the utilization of mostly glutamine to glucose but no changes in fatty acid oxidation. Interestingly, low glucose levels had less of an impact on cellular metabolism than hypoxia. A resilience to culture conditions and the capacity to utilize a broader spectrum of substrates more efficiently distinguished the highly aggressive cells from the cells representing slow-developing disease, suggesting a flexible metabolism contributes to the stem-like properties. These results indicate that adhesion to secondary sites initiates a metabolic switch in the oxidation of substrates that could support outgrowth and successful metastasis.

Mitochondrial plasticity supports proliferative outgrowth and invasion of ovarian cancer spheroids during adhesion.

Background: Ovarian cancer cells aggregate during or after exfoliation from the primary tumor to form threedimensional spheroids. Spheroid formation provides a survival advantage during peritoneal dissemination in nutrient and oxygen-depleted conditions which is accompanied by a suppressed metabolic phenotype and fragmented mitochondria. Upon arrival to their metastatic sites, spheroids adhere to peritoneal organs and transition to a more epithelial phenotype to support outgrowth and invasion. In this study, we investigated the plasticity of mitochondrial morphology, dynamics, and function upon adhesion. Methods: Using our slow-developing (MOSE-L) and fast-developing (MOSE-LTICv) ovarian cancer models, we mimicked adhesion and reoxygenation conditions by plating the spheroids onto tissue culture dishes and changing culture conditions from hypoxia and low glucose to normoxia with high glucose levels after adhesion. We used Western Blot, microscopy and Seahorse analyses to determine the plasticity of mitochondrial morphology and functions upon adhesion, and the impact on proliferation and invasion capacities. Results: Independent of culture conditions, all spheroids adhered to and began to grow onto the culture plates. While the bulk of the spheroid was unresponsive, the mitochondrial morphology in the outgrowing cells was indistinguishable from cells growing in monolayers, indicating that mitochondrial fragmentation in spheroids was indeed reversible. This was accompanied by an increase in regulators of mitobiogenesis, PGC1a, mitochondrial mass, and respiration. Reoxygenation increased migration and invasion in both cell types but only the MOSE-L responded with increased proliferation to reoxygenation. The highly aggressive phenotype of the MOSE-LTICv was characterized by a relative independence of oxygen and the preservation of higher levels of proliferation, migration and invasion even in limiting culture conditions but a higher reliance on mitophagy. Further, the outgrowth in these aggressive cells relies mostly on proliferation while the MOSE-L cells both utilize proliferation and migration to achieve outgrowth. Suppression of proliferation with cycloheximide impeded aggregation, reduced outgrowth and invasion via repression of MMP2 expression and the flattening of the spheroids. Discussion: Our studies indicate that the fragmentation of the mitochondria is reversible upon adhesion. The identification of regulatory signaling molecules and pathways of these key phenotypic alterations that occur during primary adhesion and invasion is critical for the identification of druggable targets for therapeutic intervention to prevent aggressive metastatic disease.

Quantitative biophysical metrics for rapid evaluation of ovarian cancer metastatic potential.

Ovarian cancer is routinely diagnosed long after the disease has metastasized through the fibrous submesothelium. Despite extensive research in the field linking ovarian cancer progression to increasingly poor prognosis, there are currently no validated cellular markers or hallmarks of ovarian cancer that can predict metastatic potential. To discern disease progression across a syngeneic mouse ovarian cancer progression model, here we fabricated extracellular matrix mimicking suspended fiber networks: cross-hatches of mismatch diameters for studying protrusion dynamics, aligned same diameter networks of varying interfiber spacing for studying migration, and aligned nanonets for measuring cell forces. We found that migration correlated with disease while a force-disease biphasic relationship exhibited F-actin stress fiber network dependence. However, unique to suspended fibers, coiling occurring at the tips of protrusions and not the length or breadth of protrusions displayed the strongest correlation with metastatic potential. To confirm that our findings were more broadly applicable beyond the mouse model, we repeated our studies in human ovarian cancer cell lines and found that the biophysical trends were consistent with our mouse model results. Altogether, we report complementary high throughput and high content biophysical metrics capable of identifying ovarian cancer metastatic potential on a timescale of hours.

Folate deficiency provides protection against colon carcinogenesis in DNA polymerase beta haploinsufficient mice.

Aging and DNA polymerase beta deficiency (beta-pol(+/-)) interact to accelerate the development of malignant lymphomas and adenocarcinoma and increase tumor bearing load in mice. Folate deficiency (FD) has been shown to induce DNA damage repaired via the base excision repair (BER) pathway. We anticipated that FD and BER deficiency would interact to accelerate aberrant crypt foci (ACF) formation and tumor development in beta-pol haploinsufficient animals. FD resulted in a significant increase in ACF formation in wild type (WT) animals exposed to 1,2-dimethylhydrazine, a known colon and liver carcinogen; however, FD reduced development of ACF in beta-pol haploinsufficient mice. Prolonged feeding of the FD diet resulted in advanced ACF formation and liver tumors in wild type mice. However, FD attenuated onset and progression of ACF and prevented liver tumorigenesis in beta-pol haploinsufficient mice, i.e. FD provided protection against tumorigenesis in a BER-deficient environment in all tissues where 1,2-dimethylhydrazine exerts its damage. Here we show a distinct down-regulation in DNA repair pathways, e.g. BER, nucleotide excision repair, and mismatch repair, and decline in cell proliferation, as well as an up-regulation in poly(ADP-ribose) polymerase, proapoptotic genes, and apoptosis in colons of FD beta-pol haploinsufficient mice.

Selective concentration of human cancer cells using contactless dielectrophoresis.

This work is the first to demonstrate the ability of contactless dielectrophoresis (cDEP) to isolate target cell species from a heterogeneous sample of live cells. Since all cell types have a unique molecular composition, it is expected that their dielectrophoretic (DEP) properties are also unique. cDEP is a technique developed to improve upon traditional and insulator-based DEP devices by replacing embedded metal electrodes with fluid electrode channels positioned alongside desired trapping locations. Through the placement of the fluid electrode channels and the removal of contact between the electrodes and the sample fluid, cDEP mitigates issues associated with sample/electrode contact. MCF10A, MCF7, and MDA-MB-231 human breast cells were used to represent early, intermediate, and late-staged breast cancer, respectively. Trapping frequency responses of each cell type were distinct, with the largest difference between the cells found at 20 and 30 V. MDA-MB-231 cells were successfully isolated from a population containing MCF10A and MCF7 cells at 30 V and 164 kHz. The ability to selectively concentrate cells is the key to development of biological applications using DEP. The isolation of these cells could provide a workbench for clinicians to detect transformed cells at their earliest stage, screen drug therapies prior to patient treatment, increasing the probability of success, and eliminate unsuccessful treatment options.

Selective growth inhibition of human breast cancer cells by graviola fruit extract in vitro and in vivo involving downregulation of EGFR expression.

The epidermal growth factor receptor (EGFR) is an oncogene frequently overexpressed in breast cancer (BC), and its overexpression has been associated with poor prognosis and drug resistance. EGFR is therefore a rational target for BC therapy development. This study demonstrated that a graviola fruit extract (GFE) significantly downregulated EGFR gene expression and inhibited the growth of BC cells and xenografts. GFE selectively inhibited the growth of EGFR-overexpressing human BC (MDA-MB-468) cells (IC(50) = 4.8 µg/ml) but had no effect on nontumorigenic human breast epithelial cells (MCF-10A). GFE significantly downregulated EGFR mRNA expression, arrested cell cycle in the G0/G1 phase, and induced apoptosis in MDA-MB-468 cells. In the mouse xenograft model, a 5-wk dietary treatment of GFE (200 mg/kg diet) significantly reduced the protein expression of EGFR, p-EGFR, and p-ERK in MDA-MB-468 tumors by 56%, 54%, and 32.5%, respectively. Overall, dietary GFE inhibited tumor growth, as measured by wet weight, by 32% (P < 0.01). These data showed that dietary GFE induced significant growth inhibition of MDA-MB-468 cells in vitro and in vivo through a mechanism involving the EGFR/ERK signaling pathway, suggesting that GFE may have a protective effect for women against EGFR-overexpressing BC.

Differential effects of nanosecond pulsed electric fields on cells representing progressive ovarian cancer.

Nanosecond pulsed electric fields (nsPEFs) may induce differential effects on tumor cells from different disease stages and could be suitable for treating tumors by preferentially targeting the late-stage/highly aggressive tumor cells. In this study, we investigated the nsPEF responses of mouse ovarian surface epithelial (MOSE) cells representing progressive ovarian cancer from benign to malignant stages and highly aggressive tumor-initiating-like cells. We established the cell-seeded 3D collagen scaffolds cultured with or without Nocodazole (eliminating the influence of cell proliferation on ablation outcome) to observe the ablation effects at 3 h and 24 h after treatment and compared the corresponding thresholds obtained by numerically calculated electric field distribution. The results showed that nsPEFs induced larger ablation areas with lower thresholds as the cell progress from benign, malignant to a highly aggressive phenotype. This differential effect was not affected by the different doubling times of the cells, as apparent by similar ablation induction after a synergistic treatment of nsPEFs and Nocodazole. The result suggests that nsPEFs could induce preferential ablation effects on highly aggressive and malignant ovarian cancer cells than their benign counterparts. This study provides an experimental basis for the research on killing malignant tumor cells via electrical treatments and may have clinical implications for treating tumors and preventing tumor recurrence after treatment.

Targeting Cbx3/HP1γ Induces LEF-1 and IL-21R to Promote Tumor-Infiltrating CD8 T-Cell Persistence.

Immune checkpoint blockade (ICB) relieves CD8+ T-cell exhaustion in most mutated tumors, and TCF-1 is implicated in converting progenitor exhausted cells to functional effector cells. However, identifying mechanisms that can prevent functional senescence and potentiate CD8+ T-cell persistence for ICB non-responsive and resistant tumors remains elusive. We demonstrate that targeting Cbx3/HP1γ in CD8+ T cells augments transcription initiation and chromatin remodeling leading to increased transcriptional activity at Lef1 and Il21r. LEF-1 and IL-21R are necessary for Cbx3/HP1γ-deficient CD8+ effector T cells to persist and control ovarian cancer, melanoma, and neuroblastoma in preclinical models. The enhanced persistence of Cbx3/HP1γ-deficient CD8+ T cells facilitates remodeling of the tumor chemokine/receptor landscape ensuring their optimal invasion at the expense of CD4+ Tregs. Thus, CD8+ T cells heightened effector function consequent to Cbx3/HP1γ deficiency may be distinct from functional reactivation by ICB, implicating Cbx3/HP1γ as a viable cancer T-cell-based therapy target for ICB resistant, non-responsive solid tumors.

Conjugated linoleic acid ameliorates inflammation-induced colorectal cancer in mice through activation of PPARgamma.

Conjugated linoleic acid (CLA) exerts a protective effect on experimental inflammatory bowel disease and shows promise as a chemopreventive agent against colorectal cancer (CRC) in mice, although the mechanisms by which it exerts its beneficial effects against malignancies in the gut are not completely understood. Mice lacking PPARgamma in immune and epithelial cells and PPARgamma-expressing littermates were fed either control or CLA-supplemented (1 g CLA/100 g) diets to determine the role of PPARgamma in inflammation-induced CRC. To induce tumor formation and colitis, mice were treated with azoxymethane and then challenged with 2% dextran sodium sulfate, respectively. Dietary CLA ameliorated disease activity, decreased colitis, and prevented adenocarcinoma formation in the PPARgamma-expressing floxed mice but not in the tissue-specific PPARgamma-null mice. Dietary CLA supplementation significantly decreased the percentages of macrophages in the mesenteric lymph nodes (MLN) regardless of the genotype and increased regulatory T cell numbers in MLN of PPARgamma-expressing, but not in the tissue-specific, PPARgamma-null mice. Colonic tumor necrosis factor-alpha mRNA expression was significantly suppressed in CLA-fed, PPARgamma-expressing mice. This study suggests CLA ameliorates colitis and prevents tumor formation in part through a PPARgamma-dependent mechanism.