Measuring single-cell density with high throughput enables dynamic profiling of immune cell and drug response from patient samples.

Cell density, the ratio of cell mass to volume, is an indicator of molecular crowding and therefore a fundamental determinant of cell state and function. However, existing density measurements lack the precision or throughput to quantify subtle differences in cell states, particularly in primary samples. Here we present an approach for measuring the density of 30,000 single cells per hour with a precision of 0.03% (0.0003 g/mL) by integrating fluorescence exclusion microscopy with a suspended microchannel resonator. Applying this approach to human lymphocytes, we discovered that cell density and its variation decrease as cells transition from quiescence to a proliferative state, suggesting that the level of molecular crowding decreases and becomes more regulated upon entry into the cell cycle. Using a pancreatic cancer patient-derived xenograft model, we found that the ex vivo density response of primary tumor cells to drug treatment can predict in vivo tumor growth response. Our method reveals unexpected behavior in molecular crowding during cell state transitions and suggests density as a new biomarker for functional precision medicine.

Investigative needle core biopsies for multi-omics in Glioblastoma.

Glioblastoma (GBM) is a primary brain cancer with an abysmal prognosis and few effective therapies. The ability to investigate the tumor microenvironment before and during treatment would greatly enhance both understanding of disease response and progression, as well as the delivery and impact of therapeutics. Stereotactic biopsies are a routine surgical procedure performed primarily for diagnostic histopathologic purposes. The role of investigative biopsies - tissue sampling for the purpose of understanding tumor microenvironmental responses to treatment using integrated multi-modal molecular analyses ('Multi-omics") has yet to be defined. Secondly, it is unknown whether comparatively small tissue samples from brain biopsies can yield sufficient information with such methods. Here we adapt stereotactic needle core biopsy tissue in two separate patients. In the first patient with recurrent GBM we performed highly resolved multi-omics analysis methods including single cell RNA sequencing, spatial-transcriptomics, metabolomics, proteomics, phosphoproteomics, T-cell clonotype analysis, and MHC Class I immunopeptidomics from biopsy tissue that was obtained from a single procedure. In a second patient we analyzed multi-regional core biopsies to decipher spatial and genomic variance. We also investigated the utility of stereotactic biopsies as a method for generating patient derived xenograft models in a separate patient cohort. Dataset integration across modalities showed good correspondence between spatial modalities, highlighted immune cell associated metabolic pathways and revealed poor correlation between RNA expression and the tumor MHC Class I immunopeptidome. In conclusion, stereotactic needle biopsy cores are of sufficient quality to generate multi-omics data, provide data rich insight into a patient's disease process and tumor immune microenvironment and can be of value in evaluating treatment responses. One sentence summary: Integrative multi-omics analysis of stereotactic needle core biopsies in glioblastoma.

VRK1 as a synthetic lethal target in VRK2 promoter-methylated cancers of the nervous system.

Collateral lethality occurs when loss of a gene/protein renders cancer cells dependent on its remaining paralog. Combining genome-scale CRISPR/Cas9 loss-of-function screens with RNA sequencing in over 900 cancer cell lines, we found that cancers of nervous system lineage, including adult and pediatric gliomas and neuroblastomas, required the nuclear kinase vaccinia-related kinase 1 (VRK1) for their survival in vivo. VRK1 dependency was inversely correlated with expression of its paralog VRK2. VRK2 knockout sensitized cells to VRK1 loss, and conversely, VRK2 overexpression increased cell fitness in the setting of VRK1 loss. DNA methylation of the VRK2 promoter was associated with low VRK2 expression in human neuroblastomas and adult and pediatric gliomas. Mechanistically, depletion of VRK1 reduced barrier-to-autointegration factor phosphorylation during mitosis, resulting in DNA damage and apoptosis. Together, these studies identify VRK1 as a synthetic lethal target in VRK2 promoter-methylated adult and pediatric gliomas and neuroblastomas.

FGFR-inhibitor-mediated dismissal of SWI/SNF complexes from YAP-dependent enhancers induces adaptive therapeutic resistance.

How cancer cells adapt to evade the therapeutic effects of drugs targeting oncogenic drivers is poorly understood. Here we report an epigenetic mechanism leading to the adaptive resistance of triple-negative breast cancer (TNBC) to fibroblast growth factor receptor (FGFR) inhibitors. Prolonged FGFR inhibition suppresses the function of BRG1-dependent chromatin remodelling, leading to an epigenetic state that derepresses YAP-associated enhancers. These chromatin changes induce the expression of several amino acid transporters, resulting in increased intracellular levels of specific amino acids that reactivate mTORC1. Consistent with this mechanism, addition of mTORC1 or YAP inhibitors to FGFR blockade synergistically attenuated the growth of TNBC patient-derived xenograft models. Collectively, these findings reveal a feedback loop involving an epigenetic state transition and metabolic reprogramming that leads to adaptive therapeutic resistance and provides potential therapeutic strategies to overcome this mechanism of resistance.

Suppression of Nuclear Factor-κB by Glucocorticoid Receptor Blocks Estrogen-Induced Apoptosis in Estrogen-Deprived Breast Cancer Cells.

Our clinically relevant finding is that glucocorticoids block estrogen (E2)-induced apoptosis in long-term E2-deprived (LTED) breast cancer cells. However, the mechanism remains unclear. Here, we demonstrated that E2 widely activated adipose inflammatory factors such as fatty acid desaturase 1 (FADS1), IL6, and TNFα in LTED breast cancer cells. Activation of glucocorticoid receptor (GR) by the synthetic glucocorticoid dexamethasone upregulated FADS1 and IL6, but downregulated TNFα expression. Furthermore, dexamethasone was synergistic or additive with E2 in upregulating FADS1 and IL6 expression, whereas it selectively and constantly suppressed TNFα expression induced by E2 in LTED breast cancer cells. Regarding regulation of endoplasmic reticulum stress, dexamethasone effectively blocked activation of protein kinase RNA-like endoplasmic reticulum kinase (PERK) by E2, but it had no inhibitory effects on inositol-requiring protein 1 alpha (IRE1α) expression increased by E2 Consistently, results from reverse-phase protein array (RPPA) analysis demonstrated that dexamethasone could not reverse IRE1α-mediated degradation of PI3K/Akt-associated signal pathways activated by E2 Unexpectedly, activated GR preferentially repressed nuclear factor-κB (NF-κB) DNA-binding activity and expression of NF-κB-dependent gene TNFα induced by E2, leading to the blockade of E2-induced apoptosis. Together, these data suggest that trans-suppression of NF-κB by GR in the nucleus is a fundamental mechanism thereby blocking E2-induced apoptosis in LTED breast cancer cells. This study provided an important rationale for restricting the clinical use of glucocorticoids, which will undermine the beneficial effects of E2-induced apoptosis in patients with aromatase inhibitor-resistant breast cancer.

Targeting Peroxisome Proliferator-Activated Receptor γ to Increase Estrogen-Induced Apoptosis in Estrogen-Deprived Breast Cancer Cells.

Peroxisome proliferator-activated receptor γ (PPARγ) is an important transcription factor that modulates lipid metabolism and inflammation. However, it remains unclear whether PPARγ is involved in modulation of estrogen (E2)-induced inflammation, thus affecting apoptosis of E2-deprived breast cancer cells, MCF-7:5C and MCF-7:2A. Here, we demonstrated that E2 treatment suppressed the function of PPARγ in both cell lines, although the suppressive effect in MCF-7:2A cells was delayed owing to high PPARγ expression. Activation of PPARγ by a specific agonist, pioglitazone, selectively blocked the induction of TNFα expression by E2, but did not affect other adipose inflammatory genes, such as fatty acid desaturase 1 and IL6. This suppression of TNFα expression by pioglitazone was mainly mediated by transrepression of nuclear factor-κB (NF-κB) DNA-binding activity. A novel finding was that NF-κB functions as an oxidative stress inducer in MCF-7:5C cells but an antioxidant in MCF-7:2A cells. Therefore, the NF-κB inhibitor JSH-23 displayed effects equivalent to those of pioglitazone, with complete inhibition of apoptosis in MCF-7:5C cells, but it increased E2-induced apoptosis in MCF-7:2A cells. Depletion of PPARγ by siRNA or the PPARγ antagonist T0070907 accelerated E2-induced apoptosis, with activation of NF-κB-dependent TNFα and oxidative stress. For the first time, we demonstrated that PPARγ is a growth signal and has potential to modulate NF-κB activity and oxidative stress in E2-deprived breast cancer cell lines. All of these findings suggest that anti-PPARγ therapy is a novel strategy to improve the therapeutic effects of E2-induced apoptosis in E2-deprived breast cancer.

Structural Basis of TPR-Mediated Oligomerization and Activation of Oncogenic Fusion Kinases.

The nuclear pore complex subunit TPR is found in at least five different oncogenic fusion kinases, including TPR-MET, yet how TPR fusions promote activation of kinases and their oncogenic activities remains poorly understood. Here we report the crystal structure of TPR(2-142), the MET fusion partner of oncogenic TPR-MET. TPR(2-142) contains a continuous 124-residue α helix that forms an antiparallel tetramer from two leucine zipper-containing parallel coiled coils. Remarkably, single mutations cause strikingly different conformations of the coiled coil, indicating its highly dynamic nature. We further show that fusion of TPR(2-142) to the MET intracellular domain strongly and selectively stabilizes the αG helix of the MET kinase domain, and mutations of only the TPR leucine zipper residues at the junction to MET, but not other leucine zipper residues, abolish kinase activation. Together, these results provide critical insight into the TPR structure and its ability to induce dimerization and activation of fusion kinases.

Estrogen induces apoptosis in estrogen deprivation-resistant breast cancer through stress responses as identified by global gene expression across time.

In laboratory studies, acquired resistance to long-term antihormonal therapy in breast cancer evolves through two phases over 5 y. Phase I develops within 1 y, and tumor growth occurs with either 17ß-estradiol (E(2)) or tamoxifen. Phase II resistance develops after 5 y of therapy, and tamoxifen still stimulates growth; however, E(2) paradoxically induces apoptosis. This finding is the basis for the clinical use of estrogen to treat advanced antihormone-resistant breast cancer. We interrogated E(2)-induced apoptosis by analysis of gene expression across time (2-96 h) in MCF-7 cell variants that were estrogen-dependent (WS8) or resistant to estrogen deprivation and refractory (2A) or sensitive (5C) to E(2)-induced apoptosis. We developed a method termed differential area under the curve analysis that identified genes uniquely regulated by E(2) in 5C cells compared with both WS8 and 2A cells and hence, were associated with E(2)-induced apoptosis. Estrogen signaling, endoplasmic reticulum stress (ERS), and inflammatory response genes were overrepresented among the 5C-specific genes. The identified ERS genes indicated that E(2) inhibited protein folding, translation, and fatty acid synthesis. Meanwhile, the ERS-associated apoptotic genes Bcl-2 interacting mediator of cell death (BIM; BCL2L11) and caspase-4 (CASP4), among others, were induced. Evaluation of a caspase peptide inhibitor panel showed that the CASP4 inhibitor z-LEVD-fmk was the most active at blocking E(2)-induced apoptosis. Furthermore, z-LEVD-fmk completely prevented poly (ADP-ribose) polymerase (PARP) cleavage, E(2)-inhibited growth, and apoptotic morphology. The up-regulated proinflammatory genes included IL, IFN, and arachidonic acid-related genes. Functional testing showed that arachidonic acid and E(2) interacted to superadditively induce apoptosis. Therefore, these data indicate that E(2) induced apoptosis through ERS and inflammatory responses in advanced antihormone-resistant breast cancer.

Anti-proliferative effect of LXR agonist T0901317 in ovarian carcinoma cells.

BACKGROUND: Ovarian cancer is the most common cause of cancer related death from gynecologic tumors in the United States. The insidious nature of the disease precludes early diagnosis, therefore surgical debulking and chemotherapy are considered as standard treatment modalities for advanced stages. We investigated the effect of the LXR agonist, T0901317, on ovarian cancer cell proliferation and apoptosis as a potential therapeutic agent. RESULTS: T0901317 treatment resulted in a significant (P <0.001) inhibition of cell proliferation in a time- and dose-dependent manner in CaOV3, SKOV3 and A2780 cells. Western blot analysis demonstrated an induction of p21 and p27 with a concominant reduction in phospho-RB protein levels. Cell cycle analysis demonstrated a significant (P <0.001) arrest in the G1 cell cycle phase. Significant induction of Caspase-3 and BAX gene expression occurred with treatment. Induction of apoptosis was confirmed by significant (P < 0.001) elevation of caspase activity on FACS analysis, caspase-glo assay, BAX protein induction and decreased caspase 3 precursor protein expression on Western blot analysis. LXR alpha/beta knockdown experiments did not reverse the anti-proliferative and cytotoxic effects of T0901317. CONCLUSIONS: The LXR agonist, T0901317, significantly suppresses cell proliferation and induces programmed cell death in a dose- and time-dependent manner. Our results indicate that T0901317 induces its anti-proliferative and cytotoxic effects via an LXR-independent mechanism.

The chromatin remodeling factor SRCAP modulates expression of prostate specific antigen and cellular proliferation in prostate cancer cells.

The SNF2-related CBP activator protein (SRCAP) serves as a coactivator for several nuclear receptors including the androgen receptor (AR). SRCAP is an ATPase that is the core subunit of a large multiprotein complex and was shown to incorporate the histone variant H2A.Z into nucleosomes. In this report, we demonstrate that SRCAP is expressed in the epithelium of normal prostate and in prostate carcinoma cells, and is associated with AR in the nucleus. Using transient transfection assays we demonstrate that SRCAP activates hormone-dependent transcription of the androgen responsive, prostate specific antigen (PSA)-Luciferase reporter gene in human prostate cells. The in vivo occupancy of SRCAP at the endogenous PSA promoter is demonstrated using chromatin immunoprecipitation assays. ShRNA mediated knockdown of SRCAP resulted in decreased H2A.Z binding at the enhancer region of the PSA promoter and decreased expression of PSA in prostate cancer cells. Furthermore, inhibition of SRCAP expression significantly inhibited androgen dependent prostate cancer cell growth. These data identify SRCAP as a physiologically relevant mediator of PSA expression, and demonstrate that SRCAP plays a role in prostate cancer cell proliferation.

The G protein-coupled receptor GPR30 inhibits proliferation of estrogen receptor-positive breast cancer cells.

The G protein-coupled receptor GPR30 binds 17beta-estradiol (E(2)) yet differs from classic estrogen receptors (ERalpha and ERbeta). GPR30 can mediate E(2)-induced nongenomic signaling, but its role in ERalpha-positive breast cancer remains unclear. Gene expression microarray data from five cohorts comprising 1,250 breast carcinomas showed an association between increased GPR30 expression and ERalpha-positive status. We therefore examined GPR30 in estrogenic activities in ER-positive MCF-7 breast cancer cells using G-1 and diethylstilbestrol (DES), ligands that selectively activate GPR30 and ER, respectively, and small interfering RNAs. In expression studies, E(2) and DES, but not G-1, transiently downregulated both ER and GPR30, indicating that this was ER mediated. In Ca(2+) mobilization studies, GPR30, but not ERalpha, mediated E(2)-induced Ca(2+) responses because E(2), 4-hydroxytamoxifen (activates GPR30), and G-1, but not DES, elicited cytosolic Ca(2+) increases not only in MCF-7 cells but also in ER-negative SKBr3 cells. Additionally, in MCF-7 cells, GPR30 depletion blocked E(2)-induced and G-1-induced Ca(2+) mobilization, but ERalpha depletion did not. Interestingly, GPR30-coupled Ca(2+) responses were sustained and inositol triphosphate receptor mediated in ER-positive MCF-7 cells but transitory and ryanodine receptor mediated in ER-negative SKBr3 cells. Proliferation studies involving GPR30 depletion indicated that the role of GPR30 was to promote SKBr3 cell growth but reduce MCF-7 cell growth. Supporting this, G-1 profoundly inhibited MCF-7 cell growth, potentially via p53 and p21 induction. Further, flow cytometry showed that G-1 blocked MCF-7 cell cycle progression at the G(1) phase. Thus, GPR30 antagonizes growth of ERalpha-positive breast cancer and may represent a new target to combat this disease.

beta2 Adrenoreceptor blockade attenuates the hyperinflammatory response induced by traumatic injury.

BACKGROUND: Major operative injury initiates immunologic changes that may result in a systemic inflammatory response, leading to organ dysfunction/sepsis. Catecholamines seem to be key mediators. The effects of beta(2) receptor blockade in vitro and in vivo before and after operative injury were studied to clarify their role. METHODS: In vitro studies were done in RAW 264.7 cells using epinephrine (50 micromol/L) with or without alpha(2)- and beta(2)-receptor blockade. Comparative gene expression analysis on the Toll-like receptor (TLR)-4 receptor signaling pathway was performed between RAW cells pretreated with epinephrine with subsequent lipopolysaccharide (LPS) stimulation versus LPS alone. Confirmatory studies were performed by real-time reverse transcription polymerase chain reaction (RT-PCR). Tumor necrosis factor (TNF)-alpha gene and protein expression were determined via real time RT-PCR and enzyme-linked immunosorbent assay, respectively. In vivo, Balb/C mice received no treatment nor injury (group I). Group II received operative injury and vehicle injection, group III received ICI 118,551 (beta(2)-receptor antagonist) 30 minutes before or in separate studies after operative injury. At 7 days, splenic macrophages were harvested and cytokine production was measured with or without LPS. In separate experiments, cecal ligation and puncture (CLP) was done 7 days after operation and survival determined. RESULTS: In vitro studies demonstrated that epinephrine pretreatment significantly increased TNF-alpha production with LPS stimulation (P < .05). beta(2)-Receptor blockade significantly attenuated (P < .05) LPS stimulated TNF-alpha production. MD-2, an essential coactivator of TLR-4 signaling, gene expression was significantly elevated when cells were pretreated with epinephrine before LPS exposure (P < .001). In vivo studies demonstrated a significant decrease (P < .05) in TNF-alpha and interleukin-6 production in the ICI 118,551 group. Similar findings were demonstrated measuring monocyte chemoattractant protein-1 and interferon-gamma cytokine levels (P < .05) versus no treatment. ICI 118,551 treatment 30 minutes before operation demonstrated a 31% reduction in mortality after CLP (P < .05). CONCLUSION: This study demonstrates that beta(2)-receptor blockade reduces macrophage cytokine production and improves survival showing the critical importance of catecholamines to the immunologic response in surgery.

THE PPARgamma ligand 15d-PGJ2 modulates macrophage activation after injury in a murine trauma model.

In macrophages, peroxisome proliferator-activated receptor gamma (PPARgamma) has been shown to be important for differentiation, and it serves as a negative regulator of activation. Major trauma/injury causes a dramatic host response that disrupts cellular immune homeostasis and initiates an inflammatory cascade that predisposes the injured host to subsequent infections. In prior studies using a murine trauma model consisting of femur fracture and hemorrhage, splenic macrophages from traumatized mice had significantly enhanced LPS-induced cyclooxygenase enzyme (subtype 2) and iNOS production as well as elevated levels of inflammatory cytokines at 1 week after injury compared with uninjured controls. These up-regulated cellular responses corresponded to increased mortality when animals were challenged with LPS or Candida. In the current study, we used the injury model to determine the effect of treatment of injured mice with the endogenous PPARgamma ligand 15-deoxy-Delta(12-, 14)-PGJ2 (15d-PGJ2). It was found that in vivo 15d-PGJ2 treatment significantly reduced the levels of inflammatory mediators produced by splenic macrophages 7 days after injury. The mechanism of inhibition is dependent on PPARgamma because concomitant treatment of animals with the PPARgamma antagonist GW9662 reversed the inhibitory effect of 15d-PGJ2. Endogenous PPARgamma modulated activation of LPS-induced p38 mitogen-activated protein kinase. Furthermore, treatment of injured mice with 15d-PGJ2 conferred a significant survival advantage after infectious challenge induced by cecal ligation and puncture. Thus, this PPARgamma ligands significantly attenuate the postinjury inflammatory response and improve survival after infectious challenge.