Magnetic resonance spectroscopy (MRS) or spectroscopic imaging (MRSI) enables the detection of metabolites, amino acids, and lipids, among other biomolecules, in tumors of live mouse models of ...cancer. Tumor-bearing mice are anesthetized by breathing isoflurane in a magnetic resonance (MR) scanner dedicated to small animal MR. Here we describe the overall setup and steps for measuring
H and
P MRS and
H MRSI of orthotopic breast tumor models in mice with surface coils. This protocol can be adapted to the use of volume coils to measure
H and
P MRS(I) of tumor models that grow inside the body. We address issues of animal handling, setting up the measurement, measurement options, and data analysis.
Commentary to:
ADAM17 promotes breast cancer cell malignant phenotype through EGFR-PI3K-AKT activation
Xuguang Zheng, Feng Jiang, Mark Katakowski, Zheng Gang Zhang, Qing-e Lu, Michael Chopp
Noninvasive imaging of lysosomes will be useful 1) to elucidate the role of lysosomal parameters in cancer, 2) to diagnose malignant lesions, and 3) to evaluate future lysosome-targeted anticancer ...therapies. Lysosome-specific labeling of glucosamine-bound near-infrared (NIR) fluorescent probes, IR-1 and IR-2, but not control probe IR-15 without the glucosamine moiety, was observed by fluorescence microscopy in human breast epithelial cell lines. Lysosome labeling and tumor specificity of these NIR probes were investigated by dynamic optical imaging and immunofluorescence staining in human breast tumor xenografts. IR-1 and IR-2 demonstrated faster lysosome labeling rates in highly aggressive MDA-MB-231 and MDA-MB-435 cells compared with less aggressive MCF-7 and nontumorigenic MCF-12A cells. IR-1 and IR-2, but not IR-15, accumulated in human MDA-MB-231, MDA-MB-435, and MCF-7 breast tumor xenografts in vivo. IR-2 demonstrated the highest maximum fluorescence and tumor/normal tissue ratios in all tumor models. Specific lysosome labeling from IR-2 in vivo was validated by colocalization of the NIR fluorescence with CD63 immunofluorescence in tumor sections. IR-1 and IR-2 demonstrated high lysosome-labeling ability and breast tumor-targeting specificity in vitro and in vivo. They are promising for diagnosing malignant lesions and may provide a means for evaluating and monitoring future lysosome-targeted anticancer therapies.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Cyclooxygenases (COX) are rate-limiting enzymes involved in the conversion of PLA 2 -mobilized arachidonic acid into prostaglandins and thromboxanes. COX-2 is a key mediator of inflammation during ...both physiologic
and pathologic responses to endogenous stimuli and infectious agents. Its overexpression has been detected in different cancers,
including that of the breast. Using RNA interference, we have reduced the expression of COX-2 in the highly malignant breast
cancer cell line MDA-MB-231 below detectable levels in response to interleukin-1β or 12- O -tetradecanoylphorbol-13-acetate treatment. Microarray analysis showed that COX-2 silencing resulted in the loss of mRNA expression
of several oncogenic markers, such as matrix metalloproteinase-1, chemokine (C-X-C motif) receptor 4, and interleukin-11,
which have been correlated with poor disease outcome, and in the up-regulation of antimetastatic transcripts, such as thrombospondin-1
and Epstein-Barr-Induced 3. Cells lacking COX-2 were less able to invade reconstituted extracellular matrix than parental
cells in vitro . Consistent with these changes, loss of COX-2 resulted in the abolition or the significant delay of tumor onset when the
cells were injected in the mammary fat pad of severe combined immunodeficient mice. Finally, silencing of COX-2 resulted in
the inhibition of metastasis to the lungs of severe combined immunodeficient mice after intravenous injection. These data
show that silencing of COX-2 abolishes the metastatic potential of MDA-MB-231 cells in vivo . (Mol Cancer Res 2007;5(5):435–42)
High levels of choline kinase (ChoK) expression and choline phospholipid metabolites are often associated with malignant transformation, invasion, and metastasis, particularly in breast cancer. These ...findings have led to the development of novel pharmacologic or gene therapeutic interventions for ChoK-targeted inhibition. To identify pharmacodynamic markers for the therapeutic evaluation of ChoK down-regulation, we investigated the uptake and efflux of 3Hcholine, a natural substrate of ChoK, and two other important metabolic indicators of malignancy, namely, 3Hthymidine and 3Hfluorodeoxyglucose, which measure proliferation and glucose metabolic changes, respectively, in ChoK-downregulated cells. Choline uptake in nonmalignant and malignant breast epithelial cell lines expressing graded levels of ChoK showed a ChoK-dependent uptake, retention, and efflux of 3Hcholine. Reduced proliferation observed because of ChoK down-regulation resulted in reduced 3Hthymidine uptake and incorporation into DNA within 48 hours of treatment. Reduced 3Hthymidine incorporation levels were consistent with a decreased cell cycle S-phase fraction. No change in 3Hfluorodeoxyglucose uptake was observed between ChoK-downregulated and control cells in any of the three cell lines tested. These results demonstrate the utility of radiolabeled choline or choline analogs and proliferation imaging agents as pharmacodynamic markers for ChoK-targeted therapies and suggest a ChoK-mediated mechanism for tumor sequestration of choline-based imaging agents.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
To assess the ability of a polarization transfer (PT) magnetic resonance spectroscopy (MRS) technique to improve the detection of the individual phospholipid metabolites phosphocholine (PC), ...phosphoethanolamine (PE), glycerophosphocholine (GPC), and glycerophosphoethanolamine (GPE) in vivo in breast tumor xenografts.
The adiabatic version of refocused insensitive nuclei enhanced by polarization transfer (BINEPT) MRS was tested at 9.4 Tesla in phantoms and animal models. BINEPT and pulse-acquire (PA) 31P MRS was acquired consecutively from the same orthotopic MCF-7 (n = 10) and MDA-MB-231 (n = 10) breast tumor xenografts. After in vivo MRS measurements, animals were euthanized, tumors were extracted and high resolution (HR)-MRS was performed. Signal to noise ratios (SNRs) and metabolite ratios were compared for BINEPT and PA MRS, and were also measured and compared with that from HR-MRS.
BINEPT exclusively detected metabolites with 1H-31P coupling such as PC, PE, GPC, and GPE, thereby creating a significantly improved, flat baseline because overlapping resonances from immobile and partly mobile phospholipids were removed without loss of sensitivity. GPE and GPC were more accurately detected by BINEPT in vivo, which enabled a reliable quantification of metabolite ratios such as PE/GPE and PC/GPC, which are important markers of tumor aggressiveness and treatment response.
BINEPT is advantageous over PA for detecting and quantifying the individual phospholipid metabolites PC, PE, GPC, and GPE in vivo at high magnetic field strength. As BINEPT can be used clinically, alterations in these phospholipid metabolites can be assessed in vivo for cancer diagnosis and treatment monitoring.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
In this study, we explored the relation between metastatic states vs the capacity of confined migration, amoeboid transition, and cellular stiffness. We compared across an isogenic panel of human ...breast cancer cells derived from MDA‐MB‐231 cells. It was observed that cells after lung metastasis have the fastest migration and lowest stiffness, with a significantly higher capacity to transition into an amoeboid mode. Our findings illustrate that metastasis is a selective process favoring motile and softer cells. Moreover, the observation that circulating tumor cells resemble the parental cell line, but not lung‐metastatic cells, suggests that cells with higher deformability and motility are likely selected during extravasation and colonization.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Human mammary epithelial cells (HMECs) exhibit an increase in phosphocholine (PC) and total cholinecontaining compounds, as well as a switch from high glycerophosphocholine (GPC)/low PC to low ...GPC/high PC, with progression to malignant phenotype. The treatment of human breast cancer cells with a nonsteroidal anti-inflammatory agent, indomethacin, reverted the high PC/low GPC pattern to a low PC/high GPC pattern indicative of a less malignant phenotype, supported by decreased invasion. Here, we have characterized mechanisms underlying indomethacininduced alterations in choline membrane metabolism in malignant breast cancer cells and nonmalignant HMECs labeled with 1,2-13Ccholine using 1H and 13C magnetic resonance spectroscopy. Microarray gene expression analysis was performed to understand the molecular mechanisms underlying these changes. In breast cancer cells, indomethacin treatment activated phospholipases that, combined with an increased choline phospholipid biosynthesis, led to increased GPC and decreased PC levels. However, in nonmalignant HMECs, activation of the anabolic pathway alone was detected following indomethacin treatment. Following indomethacin treatment in breast cancer cells, several candidate genes, such as interleukin 8, NGFB, CSF2, RHOB, EDN1, and JUNB, were differentially expressed, which may have contributed to changes in choline metabolism through secondary effects or signaling cascades leading to changes in enzyme activity.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Abstract
Breast cancer is one of the most commonly diagnosed cancers and the leading cause of cancer-related death among women. Triple negative breast cancer (TNBC) is a subtype of breast cancer ...characterized by the absence of estrogen receptor, progesterone receptor and HER2 expression. TNBC shows a high capacity for early metastasis and leads to worse clinical outcomes than other breast cancer subtypes due to the lack of specific therapeutic targets. We are looking to develop cancer-specific therapeutic targets for TNBC. Many types of cancer cells including TNBC cells rely on glutamine as carbon and nitrogen source to fuel unchecked growth. We screened for key genes in regulating glutamine metabolism in a panel of breast cancer cell lines. This screen identified the mRNA and protein levels of solute carrier family 38 member 3 (SLC38A3), a glutamine transporter, to be upregulated in human breast cancer cell lines, especially in TNBC cell lines. The TCGA breast cancer patient database also showed that SLC38A3 mRNA is overexpressed in invasive ductal breast carcinoma tissues, and it is even higher in TNBC relative to other breast cancer subtypes. To test the biological role of SLC38A3 protein in TNBC cells, we performed loss-of-function experiments in multiple TNBC cell lines. Silencing of SLC38A3 decreased cellular glutamate, glutamine and alanine levels. Silencing of SLC38A3 also activated apoptosis, and suppressed cell viability, migration and invasion in several TNBC cell lines. Interestingly, silencing of SLC38A3 increased the activity of glycogen synthase kinase 3-β (GSK3β) which promoted the degradation of β-catenin, leading to the decrease of the mRNA levels of epithelial-to-mesenchymal-transition (EMT)-associated transcription factors in TNBC cell lines. In summary, we showed that SLC38A3 is overexpressed in TNBC and promotes breast cancer migration and invasion via GSK3β/β-catenin/EMT pathway, which could be a novel therapeutic target for breast cancer.
Citation Format: Zheqiong Tan, Caitlin M. Tressler, Kanchan Sonkar, Kristine Glunde. Glutamine transporter SLC38A3 promotes breast cancer migration via GSK3beta/beta-catenin/EMT pathway abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB207.
The adaptability and the genomic plasticity of cancer cells, and the interaction between the tumor microenvironment and co-opted stromal cells, coupled with the ability of cancer cells to colonize ...distant organs, contribute to the frequent intractability of cancer. It is becoming increasingly evident that personalized molecular targeting is necessary for the successful treatment of this multifaceted and complex disease. Noninvasive imaging modalities such as magnetic resonance (MR), positron emission tomography (PET), and single-photon emission computed tomography (SPECT) are filling several important niches in this era of targeted molecular medicine, in applications that span from bench to bedside. In this review we focus on noninvasive magnetic resonance spectroscopy (MRS) and spectroscopic imaging (MRSI) and their roles in future personalized medicine in cancer. Diagnosis, the identification of the most effective treatment, monitoring treatment delivery, and response to treatment are some of the broad areas into which MRS techniques can be integrated to improve treatment outcomes. The development of novel probes for molecular imaging--in combination with a slew of functional imaging capabilities--makes MRS techniques, especially in combination with other imaging modalities, valuable in cancer drug discovery and basic cancer research.
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