A Novel Cartesian Plot Analysis for Fixed Monolayers That Relates Cell Phenotype to Transfer of Contents between Fibroblasts and Cancer Cells by Cell-Projection Pumping.

We recently described cell-projection pumping as a mechanism transferring cytoplasm between cells. The uptake of fibroblast cytoplasm by co-cultured SAOS-2 osteosarcoma cells changes SAOS-2 morphology and increases cell migration and proliferation, as seen by single-cell tracking and in FACS separated SAOS-2 from co-cultures. Morphological changes in SAOS-2 seen by single cell tracking are consistent with previous observations in fixed monolayers of SAOS-2 co-cultures. Notably, earlier studies with fixed co-cultures were limited by the absence of a quantitative method for identifying sub-populations of co-cultured cells, or for quantitating transfer relative to control populations of SAOS-2 or fibroblasts cultured alone. We now overcome that limitation by a novel Cartesian plot analysis that identifies individual co-cultured cells as belonging to one of five distinct cell populations, and also gives numerical measure of similarity to control cell populations. We verified the utility of the method by first confirming the previously established relationship between SAOS-2 morphology and uptake of fibroblast contents, and also demonstrated similar effects in other cancer cell lines including from melanomas, and cancers of the ovary and colon. The method was extended to examine global DNA methylation, and while there was no clear effect on SAOS-2 DNA methylation, co-cultured fibroblasts had greatly reduced DNA methylation, similar to cancer associated fibroblasts.

Phenotypic and molecular states of IDH1 mutation-induced CD24-positive glioma stem-like cells.

Mutations in IDH1 and IDH2 drive the development of gliomas. These genetic alterations promote tumor cell renewal, disrupt differentiation states, and induce stem-like properties. Understanding how this phenotypic reprogramming occurs remains an area of high interest in glioma research. Previously, we showed that IDH mutation results in the development of a CD24-positive cell population in gliomas. Here, we demonstrate that this CD24-positive population possesses striking stem-like properties at the molecular and phenotypic levels. We found that CD24 expression is associated with stem-like features in IDH-mutant tumors, a patient-derived gliomasphere model, and a neural stem cell model of IDH1-mutant glioma. In orthotopic models, CD24-positive cells display enhanced tumor initiating potency compared to CD24-negative cells. Furthermore, CD24 knockdown results in changes in cell viability, proliferation rate, and gene expression that closely resemble a CD24-negative phenotype. Our data demonstrate that induction of a CD24-positive population is one mechanism by which IDH-mutant tumors acquire stem-like properties. These findings have significant implications for our understanding of the molecular underpinnings of IDH-mutant gliomas.

Regenerative potential of prostate luminal cells revealed by single-cell analysis.

Androgen deprivation is the cornerstone of prostate cancer treatment. It results in involution of the normal gland to ~90% of its original size because of the loss of luminal cells. The prostate regenerates when androgen is restored, a process postulated to involve stem cells. Using single-cell RNA sequencing, we identified a rare luminal population in the mouse prostate that expresses stemlike genes (Sca1 + and Psca +) and a large population of differentiated cells (Nkx3.1 +, Pbsn +). In organoids and in mice, both populations contribute equally to prostate regeneration, partly through androgen-driven expression of growth factors (Nrg2, Rspo3) by mesenchymal cells acting in a paracrine fashion on luminal cells. Analysis of human prostate tissue revealed similar differentiated and stemlike luminal subpopulations that likewise acquire enhanced regenerative potential after androgen ablation. We propose that prostate regeneration is driven by nearly all persisting luminal cells, not just by rare stem cells.

Potential Hydrodynamic Cytoplasmic Transfer between Mammalian Cells: Cell-Projection Pumping.

We earlier reported cytoplasmic fluorescence exchange between cultured human fibroblasts (Fibs) and malignant cells (MCs). Others report similar transfer via either tunneling nanotubes (TNTs) or shed membrane vesicles, and this changes the phenotype of recipient cells. Our time-lapse microscopy showed most exchange was from Fibs into MCs, with less in the reverse direction. Although TNTs were seen, we were surprised transfer was not via TNTs but was instead via fine and often branching cell projections that defied direct visual resolution because of their size and rapid movement. Their structure was revealed nonetheless by their organellar cargo and the grooves they formed indenting MCs, which was consistent with holotomography. Discrete, rapid, and highly localized transfer events evidenced against a role for shed vesicles. Transfer coincided with rapid retraction of the cell projections, suggesting a hydrodynamic mechanism. Increased hydrodynamic pressure in retracting cell projections normally returns cytoplasm to the cell body. We hypothesize "cell-projection pumping" (CPP), in which cytoplasm in retracting cell projections partially equilibrates into adjacent recipient cells via microfusions that form temporary intercellular cytoplasmic continuities. We tested plausibility for CPP by combined mathematical modeling, comparison of predictions from the model with experimental results, and then computer simulations based on experimental data. The mathematical model predicted preferential CPP into cells with lower cell stiffness, expected from equilibration of pressure toward least resistance. Predictions from the model were satisfied when Fibs were cocultured with MCs and fluorescence exchange was related to cell stiffness by atomic force microscopy. When transfer into 5000 simulated recipient MCs or Fibs was studied in computer simulations, inputting experimental cell stiffness and donor cell fluorescence values generated transfers to simulated recipient cells similar to those seen by experiment. We propose CPP as a potentially novel mechanism in mammalian intercellular cytoplasmic transfer and communication.

A Secondary Mutation in BRAF Confers Resistance to RAF Inhibition in a BRAFV600E-Mutant Brain Tumor.

BRAFV600E hyperactivates ERK and signals as a RAF inhibitor-sensitive monomer. Although RAF inhibitors can produce impressive clinical responses in patients with mutant BRAF tumors, the mechanisms of resistance to these drugs are incompletely characterized. Here, we report a complete response followed by clinical progression in a patient with a BRAFV600E-mutant brain tumor treated with dabrafenib. Whole-exome sequencing revealed a secondary BRAFL514V mutation at progression that was not present in the pretreatment tumor. Expressing BRAFV600E/L514V induces ERK signaling, promotes RAF dimer formation, and is sufficient to confer resistance to dabrafenib. Newer RAF dimer inhibitors and an ERK inhibitor are effective against BRAFL514V-mediated resistance. Collectively, our results validate a novel biochemical mechanism of RAF inhibitor resistance mediated by a secondary mutation, emphasizing that, like driver mutations in cancer, the spectrum of mutations that drive resistance to targeted therapy are heterogeneous and perhaps emerge with a lineage-specific prevalence.Significance: In contrast to receptor tyrosine kinases, in which secondary mutations are often responsible for acquired resistance, second-site mutations in BRAF have not been validated in clinically acquired resistance to RAF inhibitors. We demonstrate a secondary mutation in BRAF (V600E/L514V) following progression on dabrafenib and confirm functionally that this mutation is responsible for resistance. Cancer Discov; 8(9); 1130-41. ©2018 AACR.See related commentary by Romano and Kwong, p. 1064This article is highlighted in the In This Issue feature, p. 1047.

PDLIM7 and CDH18 regulate the turnover of MDM2 during CDK4/6 inhibitor therapy-induced senescence.

CDK4/6 inhibitors are being used to treat a variety of human malignancies. In well-differentiated and dedifferentiated liposarcoma their clinical promise is associated with their ability to downregulate the MDM2 protein. The downregulation of MDM2 following treatment with CDK4/6 inhibitors also induces many cultured tumor cell lines derived from different types of malignancies to progress from quiescence into senescence. Here we used cultured human cell lines and defined a role for PDLIM7 and CDH18, regulating MDM2 protein in CDK4/6 inhibitor-treated cells. Materials from our previous phase II trials with palbociclib were then used to demonstrate that expression of CDH18 protein was associated with response, measured as both progression-free survival and overall survival. This supports the hypothesis that the biologic transition from quiescence to senescence has clinical relevance for this class of drugs.

Direct engagement of the PI3K pathway by mutant KIT dominates oncogenic signaling in gastrointestinal stromal tumor.

Gastrointestinal stromal tumors (GISTs) predominantly harbor activating mutations in the receptor tyrosine kinase KIT. To genetically dissect in vivo the requirement of different signal transduction pathways emanating from KIT for tumorigenesis, the oncogenic KitV558Δ mutation was combined with point mutations abrogating specific phosphorylation sites on KIT. Compared with single-mutant KitV558Δ/+ mice, double-mutant KitV558Δ;Y567F/Y567F knock-in mice lacking the SRC family kinase-binding site on KIT (pY567) exhibited attenuated MAPK signaling and tumor growth. Surprisingly, abrogation of the PI3K-binding site (pY719) in KitV558Δ;Y719F/Y719F mice prevented GIST development, although the interstitial cells of Cajal (ICC), the cells of origin of GIST, were normal. Pharmacologic inhibition of the PI3K pathway in tumor-bearing KitV558Δ/+ mice with the dual PI3K/mTOR inhibitor voxtalisib, the pan-PI3K inhibitor pilaralisib, and the PI3K-alpha-restricted inhibitor alpelisib each diminished tumor proliferation. The addition of the MEK inhibitor PD-325901 or binimetinib further decreased downstream KIT signaling. Moreover, combining PI3K and MEK inhibition was effective against imatinib-resistant KitV558Δ;T669I/+ tumors.

Iron deposition is associated with differential macrophage infiltration and therapeutic response to iron chelation in prostate cancer.

Immune cells such as macrophages are drivers and biomarkers of most cancers. Scoring macrophage infiltration in tumor tissue provides a prognostic assessment that is correlated with disease outcome and therapeutic response, but generally requires invasive biopsy. Routine detection of hemosiderin iron aggregates in macrophages in other settings histologically and in vivo by MRI suggests that similar assessments in cancer can bridge a gap in our ability to assess tumor macrophage infiltration. Quantitative histological and in vivo MRI assessments of non-heme cellular iron revealed that preclinical prostate tumor models could be differentiated according to hemosiderin iron accumulation-both in tumors and systemically. Monitoring cellular iron levels during "off-label" administration of the FDA-approved iron chelator deferiprone evidenced significant reductions in tumor size without extensive perturbation to these iron deposits. Spatial profiling of the iron-laden infiltrates further demonstrated that higher numbers of infiltrating macrophage iron deposits was associated with lower anti-tumor chelation therapy response. Imaging macrophages according to their innate iron status provides a new phenotypic window into the immune tumor landscape and reveals a prognostic biomarker associated with macrophage infiltration and therapeutic outcome.

Squamous cell carcinoma-related oncogene (SCCRO) neddylates Cul3 protein to selectively promote midbody localization and activity of Cul3KLHL21 protein complex during abscission.

Squamous cell carcinoma-related oncogene (SCCRO)/DCUN1D1, a component of the neddylation E3 complex, regulates the activity of the cullin-RING-ligase type of ubiquitination E3s by promoting neddylation of cullin family members. Studies have shown that SCCRO regulates proliferation in vitro and in vivo Here we show that inactivation of SCCRO results in prolonged mitotic time because of delayed and/or failed abscission. The effects of SCCRO on abscission involve its role in neddylation and localization of Cul3 to the midbody. The Cul3 adaptor KLHL21 mediates the effects of SCCRO on abscission, as it fails to localize to the midbody in SCCRO-deficient cells during abscission, and its inactivation resulted in phenotypic changes identical to SCCRO inactivation. Ubiquitination-promoted turnover of Aurora B at the midbody was deficient in SCCRO- and KLHL21-deficient cells, suggesting that it is the target of Cul3KLHL21 at the midbody. Correction of abscission delays in SCCRO-deficient cells with addition of an Aurora B inhibitor at the midbody stage suggests that Aurora B is the target of SCCRO-promoted Cul3KLHL21 activity. The activity of other Cul3-anchored complexes, including Cul3KLHL9/KLHL13, was intact in SCCRO-deficient cells, suggesting that SCCRO selectively, rather than collectively, neddylates cullins in vivo Combined, these findings support a model in which the SCCRO, substrate, and substrate adaptors cooperatively provide tight control of neddylation and cullin-RING-ligase activity in vivo.

Targeting acid sphingomyelinase with anti-angiogenic chemotherapy.

Despite great promise, combining anti-angiogenic and conventional anti-cancer drugs has produced limited therapeutic benefit in clinical trials, presumably because mechanisms of anti-angiogenic tissue response remain only partially understood. Here we define a new paradigm, in which anti-angiogenic drugs can be used to chemosensitize tumors by targeting the endothelial acid sphingomyelinase (ASMase) signal transduction pathway. We demonstrate that paclitaxel and etoposide, but not cisplatin, confer ASMase-mediated endothelial injury within minutes. This rapid reaction is required for human HCT-116 colon cancer xenograft complete response and growth delay. Whereas VEGF inhibits ASMase, anti-VEGFR2 antibodies de-repress ASMase, enhancing endothelial apoptosis and drug-induced tumor response in asmase+/+, but not in asmase-/-, hosts. Such chemosensitization occurs only if the anti-angiogenic drug is delivered 1-2h before chemotherapy, but at no other time prior to or post chemotherapy. Our studies suggest that precisely-timed administration of anti-angiogenic drugs in combination with ASMase-targeting anti-cancer drugs is likely to optimize anti-tumor effects of systemic chemotherapy. This strategy warrants evaluation in future clinical trials.

A novel cell-stiffness-fingerprinting analysis by scanning atomic force microscopy: comparison of fibroblasts and diverse cancer cell lines.

Differing stimuli affect cell stiffness while cancer metastasis is associated with reduced cell stiffness. Cell stiffness determined by atomic force microscopy has been limited by measurement over nuclei to avoid spurious substratum effects in thin cytoplasmic domains, and we sought to develop a more complete approach including cytoplasmic areas. Ninety µm square fields were recorded from ten separate sites of cultured human dermal fibroblasts (HDF) and three sites each for melanoma (MM39, WM175, and MeIRMu), osteosarcoma (SAOS-2 and U2OS), and ovarian carcinoma (COLO316 and PEO4) cell lines, each site providing 1024 measurements as 32 × 32 square grids. Stiffness recorded below 0.8 µm height was occasionally influenced by substratum, so only stiffness recorded above 0.8 µm was analysed, but all sites were included for height and volume analysis. COLO316 had the lowest cell height and volume, followed by HDF (p < 0.0001) and then PEO4, SAOS-2, MeIRMu, WM175, U2OS, and MM39. HDF were more stiff than all other cells (p < 0.0001), while in descending order of stiffness were PEO4, COLO316, WM175, SAOS-2, U2OS, MM39, and MeIRMu (p < 0.02). Stiffness fingerprints comprised scattergrams of stiffness values plotted against the height at which each stiffness value was recorded and appeared unique for each cell type studied, although in most cases the overall form of fingerprints was similar, with maximum stiffness at low height measurements and a second lower peak occurring at high-height levels. We suggest that our stiffness-fingerprint analytical method provides a more nuanced description than previously reported and will facilitate study of the stiffness response to cell stimulation.

Distinct but Spatially Overlapping Intestinal Niches for Vancomycin-Resistant Enterococcus faecium and Carbapenem-Resistant Klebsiella pneumoniae.

Antibiotic resistance among enterococci and γ-proteobacteria is an increasing problem in healthcare settings. Dense colonization of the gut by antibiotic-resistant bacteria facilitates their spread between patients and also leads to bloodstream and other systemic infections. Antibiotic-mediated destruction of the intestinal microbiota and consequent loss of colonization resistance are critical factors leading to persistence and spread of antibiotic-resistant bacteria. The mechanisms underlying microbiota-mediated colonization resistance remain incompletely defined and are likely distinct for different antibiotic-resistant bacterial species. It is unclear whether enterococci or γ-proteobacteria, upon expanding to high density in the gut, confer colonization resistance against competing bacterial species. Herein, we demonstrate that dense intestinal colonization with vancomycin-resistant Enterococcus faecium (VRE) does not reduce in vivo growth of carbapenem-resistant Klebsiella pneumoniae. Reciprocally, K. pneumoniae does not impair intestinal colonization by VRE. In contrast, transplantation of a diverse fecal microbiota eliminates both VRE and K. pneumoniae from the gut. Fluorescence in situ hybridization demonstrates that VRE and K. pneumoniae localize to the same regions in the colon but differ with respect to stimulation and invasion of the colonic mucus layer. While VRE and K. pneumoniae occupy the same three-dimensional space within the gut lumen, their independent growth and persistence in the gut suggests that they reside in distinct niches that satisfy their specific in vivo metabolic needs.

CHZ868, a Type II JAK2 Inhibitor, Reverses Type I JAK Inhibitor Persistence and Demonstrates Efficacy in Myeloproliferative Neoplasms.

Although clinically tested JAK inhibitors reduce splenomegaly and systemic symptoms, molecular responses are not observed in most myeloproliferative neoplasm (MPN) patients. We previously demonstrated that MPN cells become persistent to type I JAK inhibitors that bind the active conformation of JAK2. We investigated whether CHZ868, a type II JAK inhibitor, would demonstrate activity in JAK inhibitor persistent cells, murine MPN models, and MPN patient samples. JAK2 and MPL mutant cell lines were sensitive to CHZ868, including type I JAK inhibitor persistent cells. CHZ868 showed significant activity in murine MPN models and induced reductions in mutant allele burden not observed with type I JAK inhibitors. These data demonstrate that type II JAK inhibition is a viable therapeutic approach for MPN patients.

The IL-6/JAK/Stat3 feed-forward loop drives tumorigenesis and metastasis.

We have investigated the importance of interleukin-6 (IL-6) in promoting tumor growth and metastasis. In human primary breast cancers, increased levels of IL-6 were found at the tumor leading edge and positively correlated with advanced stage, suggesting a mechanistic link between tumor cell production of IL-6 and invasion. In support of this hypothesis, we showed that the IL-6/Janus kinase (JAK)/signal transducer and activator of transcription 3 (Stat3) pathway drives tumor progression through the stroma and metastatic niche. Overexpression of IL-6 in tumor cell lines promoted myeloid cell recruitment, angiogenesis, and induced metastases. We demonstrated the therapeutic potential of interrupting this pathway with IL-6 receptor blockade or by inhibiting its downstream effectors JAK1/2 or Stat3. These clinically relevant interventions did not inhibit tumor cell proliferation in vitro but had profound effects in vivo on tumor progression, interfering broadly with tumor-supportive stromal functions, including angiogenesis, fibroblast infiltration, and myeloid suppressor cell recruitment in both the tumor and pre-metastatic niche. This study provides the first evidence for IL-6 expression at the leading edge of invasive human breast tumors and demonstrates mechanistically that IL-6/JAK/Stat3 signaling plays a critical and pharmacologically targetable role in orchestrating the composition of the tumor microenvironment that promotes growth, invasion, and metastasis.

GLP-1 receptor agonists and the thyroid: C-cell effects in mice are mediated via the GLP-1 receptor and not associated with RET activation.

Liraglutide and exenatide are glucagon-like peptide receptor (GLP-1R) agonists used in the treatment of type 2 diabetes. Both molecules have been associated with the development of thyroid C-cell tumors after lifetime exposure in rodents. Previously, it has been reported that these tumors are preceded by increased plasma calcitonin and C-cell hyperplasia. We can now document that the murine C-cell effects are mediated via GLP-1R. Thus, 13 wk of continuous exposure to GLP-1R agonists was associated with marked increases in plasma calcitonin and in the incidence of C-cell hyperplasia in wild-type mice. In contrast, similar effects were not seen in GLP-1R knockout mice. Human C-cell cancer is often caused by activating mutations in the rearranged-during-transfection (RET) protooncogene. We developed an immunohistochemical method to assess RET activation in tissues. Liraglutide dosing to mice was not found to activate RET. Further evaluation of the signaling pathways demonstrated that liraglutide increased ribosomal S6, but not MAPK kinase, phosphorylation. These observations are consistent with effects of GLP-1R agonists on rodent C cells being mediated via mammalian target of rapamycin activation in a RET- and MAPK-independent manner.

Deletion of Adam10 in endothelial cells leads to defects in organ-specific vascular structures.

During vertebrate angiogenesis, Notch regulates the cell-fate decision between vascular tip cells versus stalk cells. Canonical Notch signaling depends on sequential proteolytic events, whereby interaction of Notch with membrane-anchored ligands triggers proteolytic processing, first by Adam10 and then presenilins. This liberates the Notch intracellular domain, allowing it to enter the nucleus and activate Notch-dependent genes. Here we report that conditional inactivation of Adam10 in endothelial cells (A10ΔEC) recapitulates the increased branching and density of the retinal vasculature that is also caused by interfering with Notch signaling. Moreover, A10ΔEC mice have additional vascular abnormalities, including aberrant subcapsular hepatic veins, enlarged glomeruli, intestinal polyps containing endothelial cell masses, abnormal endochondral ossification, leading to stunted long bone growth and increased pathologic neovascularization following oxygen-induced retinopathy. Our findings support a model in which Adam10 is a crucial regulator of endothelial cell-fate decisions, most likely because of its essential role in canonical Notch signaling.

Immunohistochemical assessment of signal transduction and cell-cycle networks in neural tumors.

The ability to detect transient changes in molecular networks lies at the heart of cancer biology research. This is especially apparent during tumorigenesis, where initiating mutations typically affect mitogens and cell-cycle molecules such as PDGF or retinoblastoma protein (Rb). One of the primary consequences of such processes is the inappropriate stimulation of downstream targets, normally through posttranslational modification. Immunohistochemistry (IHC) provides an important tool for assessing such changes in situ, permitting different aspects of tumor biology to be examined as a tissue undergoes transformation. Nevertheless, this can be difficult to achieve, particularly in complex environments like the brain. Here, we provide the automated methodology we have employed for the successful detection of phosphorylation of S6 ribosomal protein (S6-RP) and the retinoblastoma protein (Rb) in response to PDGF stimulation in a mouse model of glial brain tumor development.

Double in situ detection of sonic hedgehog mRNA and pMAPK protein in examining the cell proliferation signaling pathway in mouse embryo.

Double in situ detection of RNA molecules and proteins in tissue sections is not trivial. A successful experiment heavily depends on the preparation of the tissue as well as the quality of the probes and antibodies. Detection of two or more molecular markers also requires reagents and experimental conditions that will preserve authenticity (accuracy) of the single staining patterns. Here, we describe in detail the protocols used to detect sonic hedgehog (Shh) mRNA by in situ hybridization and immunofluorescence staining for phosphorylated mitogen-activated protein kinase (pMAPK) in the same mouse embryonic tissue sections. In addition to protocols for manual immuno-staining, we provide data from automated machine-based staining protocols and highly recommend it to achieve strong signal and reproducible results.

Imatinib upregulates compensatory integrin signaling in a mouse model of gastrointestinal stromal tumor and is more effective when combined with dasatinib.

Activating mutations in the Kit receptor tyrosine kinase are associated with gastrointestinal stromal tumor (GIST). Imatinib inhibits Kit and is front-line therapy for GIST. However, imatinib most often elicits a partial response or stable disease, and most GIST patients who initially respond to imatinib eventually acquire resistance. Thus, improved treatment strategies for GIST are needed. We investigated the role of Src family kinases (SFK) in tumorigenesis in a mouse model of human GIST. The SFKs Src and Lyn were active in GIST, and surprisingly, imatinib treatment stimulated their phosphorylation/activation. We show that integrin signaling activates focal adhesion kinase and, consequently, SFKs in GIST and that imatinib enhances integrin signaling, implying a role for the extracellular matrix and integrin signaling in tumor maintenance and imatinib resistance. Dasatinib, an inhibitor of SFKs and Kit, inhibited SFK and focal adhesion kinase activation in GIST but also inhibited Kit and Kit-dependent downstream signaling pathways including phosphoinositide 3-kinase and mitogen-activated protein kinase, but not signal transducer and activator of transcription (STAT) signaling. Whereas dasatinib and imatinib alone both produced a minimal histopathologic response, combination therapy improved their efficacy, leading to increased necrosis in GIST. These results highlight the importance of SFK and STAT signaling in GIST and suggest that the clinical efficacy of imatinib may be limited by the stimulation of integrin signaling.