3D chromatin remodeling potentiates transcriptional programs driving cell invasion.

The contribution of deregulated chromatin architecture, including topologically associated domains (TADs), to cancer progression remains ambiguous. CCCTC-binding factor (CTCF) is a central regulator of higher-order chromatin structure that undergoes copy number loss in over half of all breast cancers, but the impact of this defect on epigenetic programming and chromatin architecture remains unclear. We find that under physiological conditions, CTCF organizes subTADs to limit the expression of oncogenic pathways, including phosphatidylinositol 3-kinase (PI3K) and cell adhesion networks. Loss of a single CTCF allele potentiates cell invasion through compromised chromatin insulation and a reorganization of chromatin architecture and histone programming that facilitates de novo promoter-enhancer contacts. However, this change in the higher-order chromatin landscape leads to a vulnerability to inhibitors of mTOR. These data support a model whereby subTAD reorganization drives both modification of histones at de novo enhancer-promoter contacts and transcriptional up-regulation of oncogenic transcriptional networks.

Evaluation of a 'plug and play' nanoflow liquid chromatography system for MS-based proteomic characterization of clinical FFPE specimens.

INTRODUCTION: Proteomic analysis of formalin-fixed paraffin-embedded (FFPE) tumor tissue specimens has gained interest in the last 5 years due to technological advances and improved sample collection, as well as biobanking for clinical trials. The real-world implementation of clinical proteomics to these specimens, however, is hampered by tedious sample preparation steps and long instrument acquisition times. AREAS COVERED: To advance the translation of quantitative proteomics into the clinic, we are comparing the performance of the leading commercial nanoflow liquid chromatography (nLC) system (based on literature reviews), the Easy-nLC 1200 (Thermo Fisher Scientific, Waltham, MA, U.S.A.), to the Evosep One HPLC (Evosep Biosystems, Odense, Denmark). We measured FFPE-tissue digests from 21 biological replicates with a similar gradient on both of the LC systems while keeping the on-column amount (1 µg total protein) and the single-shot data-dependent acquisition-based MS/MS method constant. EXPERT OPINION: Overall, the Evosep One facilitates robust and sensitive high-throughput sample acquisition, making it suitable for clinical MS. We found the Evosep One to be a useful platform for positioning mass spectrometry-based proteomics in the clinical setting. The clinical application of nLC/MS will inform clinical decision-making in oncology and other diseases.

A Non-Hazardous Deparaffinization Protocol Enables Quantitative Proteomics of Core Needle Biopsy-Sized Formalin-Fixed and Paraffin-Embedded (FFPE) Tissue Specimens.

Most human tumor tissues that are obtained for pathology and diagnostic purposes are formalin-fixed and paraffin-embedded (FFPE). To perform quantitative proteomics of FFPE samples, paraffin has to be removed and formalin-induced crosslinks have to be reversed prior to proteolytic digestion. A central component of almost all deparaffinization protocols is xylene, a toxic and highly flammable solvent that has been reported to negatively affect protein extraction and quantitative proteome analysis. Here, we present a 'green' xylene-free protocol for accelerated sample preparation of FFPE tissues based on paraffin-removal with hot water. Combined with tissue homogenization using disposable micropestles and a modified protein aggregation capture (PAC) digestion protocol, our workflow enables streamlined and reproducible quantitative proteomic profiling of FFPE tissue. Label-free quantitation of FFPE cores from human ductal breast carcinoma in situ (DCIS) xenografts with a volume of only 0.79 mm3 showed a high correlation between replicates (r2 = 0.992) with a median %CV of 16.9%. Importantly, this small volume is already compatible with tissue micro array (TMA) cores and core needle biopsies, while our results and its ease-of-use indicate that further downsizing is feasible. Finally, our FFPE workflow does not require costly equipment and can be established in every standard clinical laboratory.

Precise Quantitation of PTEN by Immuno-MRM: A Tool To Resolve the Breast Cancer Biomarker Controversy.

The tumor suppressor PTEN is the main negative regulator of PI3K/AKT/mTOR signaling and is commonly found downregulated in breast cancer (BC). Conflicting data from conventional immunoassays such as immunohistochemistry (IHC) has sparked controversy about PTEN's role as a prognostic and predictive biomarker in BC, which can be largely attributed to the lack of specificity, sensitivity, and interlaboratory standardization. Here, we present a fully standardized, highly sensitive, robust microflow immuno-MRM (iMRM) assay that enables precise quantitation of PTEN concentrations in cells and fresh frozen (FF) and formalin-fixed paraffin-embedded (FFPE) tissues, down to 0.1 fmol/10 µg of extracted protein, with high interday and intraday precision (CV 6.3%). PTEN protein levels in BC PDX samples that were determined by iMRM correlate well with semiquantitative IHC and WB data. iMRM, however, allowed the precise quantitation of PTEN-even in samples that were deemed to be PTEN negative by IHC or western blot (WB)-while requiring substantially less tumor tissue than WB. This is particularly relevant because the extent of PTEN downregulation in tumors has been shown to correlate with severity. Our standardized and robust workflow includes an 11 min microflow LC-MRM analysis on a triple-quadrupole MS and thus provides a much needed tool for the study of PTEN as a potential biomarker for BC.

A multiplexed, automated immuno-matrix assisted laser desorption/ionization mass spectrometry assay for simultaneous and precise quantitation of PTEN and p110α in cell lines and tumor tissues.

The PI3-kinase/AKT/mTOR pathway plays a central role in cancer signaling. While p110α is the catalytic α-subunit of PI3-kinase and a major drug target, PTEN is the main negative regulator of the PI3-kinase/AKT/mTOR pathway. PTEN is often down-regulated in cancer, and there are conflicting data on PTEN's role as breast cancer biomarker. PTEN and p110α protein expression in tumors is commonly analyzed by immunohistochemistry, which suffers from poor multiplexing capacity, poor standardization, and antibody crossreactivity, and which provides only semi-quantitative data. Here, we present an automated, and standardized immuno-matrix-assisted laser desorption/ionization mass spectrometry (iMALDI) assay that allows precise and multiplexed quantitation of PTEN and p110α concentrations, without the limitations of immunohistochemistry. Our iMALDI assay only requires a low-cost benchtop MALDI-TOF mass spectrometer, which simplifies clinical translation. We validated our assay's precision and accuracy, with simultaneous enrichment of both target proteins not significantly affecting the precision and accuracy of the quantitation when compared to the PTEN- and p110α-singleplex iMALDI assays (<15% difference). The multiplexed assay's linear range is from 0.6-20 fmol with accuracies of 90-112% for both target proteins, and the assay is free of matrix-related interferences. The inter-day reproducibility over 5-days was high, with an overall CV of 9%. PTEN and p110α protein concentrations can be quantified down to 1.4 fmol and 0.6 fmol per 10 µg of total tumor protein, respectively, in various tumor tissue samples, including fresh-frozen breast tumors and colorectal cancer liver metastases, and patient-derived xenograft (PDX) tumors.

Using Two Peptide Isotopologues as Internal Standards for the Streamlined Quantification of Low-Abundance Proteins by Immuno-MRM and Immuno-MALDI.

Mass spectrometry (MS), particularly targeted proteomics, is increasingly being used for quantifying specific proteins and peptides in clinical specimens. The coupling of immuno-enrichment of proteotypic peptides with MS [e.g., immuno-multiple reaction monitoring (MRM) and immuno-matrix-assisted laser desorption ionization (MALDI)] enables the development of highly sensitive and specific assays for low-abundance signaling proteins. By incorporating stable isotope-labeled standards, these workflows allow the determination of endogenous protein concentrations. This is typically achieved through external calibration, often using surrogate matrices, which has inherent limitations for the analysis of clinical specimens as there are often substantial variations in the sample matrix, and sample amounts are typically limited. We have previously introduced the use of two peptide isotopologues for generating external calibration curves in plasma. Here, we present a two-point internal calibration (2-PIC) strategy using two isotopologues for immuno-MS assays and demonstrate its flexibility and robustness. Quantification of the tumor suppressor PTEN in Colo-205 cells by immuno-MRM and immuno-MALDI using 2-PIC and external calibration yielded very similar results (relative standard deviation between 2-PIC and external calibration: 4.9% for immuno-MRM; 1.1% for immuno-MALDI), without the need for a surrogate matrix or additional patient material for calibration, while concurrently reducing the instrument time and cost. Although our PTEN immuno-MRM and immuno-MALDI assays can be considered to be orthogonal as they utilized entirely different sample preparation and MS analysis workflows, targeted different PTEN peptides, and were performed in different laboratories, the endogenous Colo-205 PTEN levels determined with 2-PIC showed a good correlation (r2 = 0.9966) and good agreement (0.48 ± 0.01 and 0.29 ± 0.02 fmol/µg of total protein) between immuno-MRM and immuno-MALDI.

Breast cancer mammospheres secrete Adrenomedullin to induce lipolysis and browning of adjacent adipocytes.

BACKGROUND: Cancer cells cooperate with cells that compose their environment to promote tumor growth and invasion. Among them, adipocytes provide lipids used as a source of energy by cancer cells and adipokines that contribute to tumor expansion. Mechanisms supporting the dynamic interactions between cancer cells and stromal adipocytes, however, remain unclear. METHODS: We set-up a co-culture model with breast cancer cells grown in 3D as mammospheres and human adipocytes to accurately recapitulate intrinsic features of tumors, such as hypoxia and cancer cell-adipocytes interactions. RESULTS: Herein, we observed that the lipid droplets' size was reduced in adipocytes adjacent to the mammospheres, mimicking adipocyte morphology on histological sections. We showed that the uncoupling protein UCP1 was expressed in adipocytes close to tumor cells on breast cancer histological sections as well as in adipocytes in contact with the mammospheres. Mammospheres produced adrenomedullin (ADM), a multifactorial hypoxia-inducible peptide while ADM receptors were detected in adipocytes. Stimulation of adipocytes with ADM promoted UCP1 expression and increased HSL phosphorylation, which activated lipolysis. Invalidation of ADM in breast cancer cells dramatically reduced UCP1 expression in adipocytes. CONCLUSIONS: Breast tumor cells secreted ADM that modified cancer-associated adipocytes through paracrine signaling, leading to metabolic changes and delipidation. Hence, ADM appears to be crucial in controlling the interactions between cancer cells and adipocytes and represents an excellent target to hinder them.

Immuno-Matrix-Assisted Laser Desorption/Ionization Assays for Quantifying AKT1 and AKT2 in Breast and Colorectal Cancer Cell Lines and Tumors.

The phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mechanistic target of rapamycin (mTOR) pathway is one of the most commonly dysregulated signaling pathways that is linked to cancer development and progression, and its quantitative protein analysis holds the promise to facilitate patient stratification for targeted therapies. Whereas immunohistochemistry (IHC) and immunoassays are routinely used for clinical analysis of signaling pathways, mass spectrometry-based approaches such as liquid chromatography/electrospray ionization multiple reaction monitoring mass spectrometry (LC/ESI-MRM-MS) are more commonly used in clinical research. Both technologies have certain disadvantages, namely, the nonspecificity of IHC and immunoassays, and potentially long analysis times per sample of LC/ESI-MRM-MS. To create a robust, fast, and sensitive protein quantification tool, we developed immuno-matrix-assisted laser desorption/ionization (iMALDI) assays with automated liquid handling. The assays are able to quantify AKT1 and AKT2 from breast cancer and colon cancer cell lines and flash-frozen tumor lysates with a linear range of 0.05-2.0 fmol/µg of total lysate protein and with coefficients of variation < 15%. Compared to other mass spectrometric methods, the developed assays require less sample per analysis-only 25 µg of total protein-and are therefore suitable for analysis of needle biopsies. Furthermore, the presented iMALDI technique is the first MS-based method for absolute quantitation of AKT peptides from cancer tissues. This study demonstrates the suitability of iMALDI for low limit-of-detection and reproducible quantitation of signaling pathway members using a benchtop MALDI mass spectrometer within approximately 6-7 h.

The identification of challenges in tissue collection for biomarker studies: the Q-CROC-03 neoadjuvant breast cancer translational trial experience.

One of the major challenges in biomarker development is the collection of tumor tissue of adequate quality for analysis. A prospective clinical trial was initiated to collect tissues from triple negative breast cancers prior to and after neoadjuvant chemotherapy in order to study the mechanisms of chemoresistance. Sixty patients had pre-chemotherapy biopsies performed by either a surgeon or a radiologist, while those with residual tumor after chemotherapy had research-only biopsies and/or surgical samples collected in liquid nitrogen, RNA-later and formalin. We examined each core for tumor cellularity, stromal content, and necrosis after which, RNA and DNA extraction was performed. We found that biopsies collected with ultrasound guidance were more likely to contain tumor than those collected by the surgeon. Patient reluctance to undergo research-only biopsies after chemotherapy was not a problem. Pre-chemotherapy tumor biopsies frequently did not contain any tumor cells (15%) or did not have ≥50% tumor content (63%). Indeed, 50% of patients had at least 2 pre-chemotherapy core biopsies with <50% tumor content. After chemotherapy, 30% of biopsy or surgical samples in patients with incomplete response did not contain any tumor. Finally, RNA-later not only made histopathological assessment of tumor content difficult, but yielded less DNA than fresh snap frozen samples. We recommend that high-quality tissue procurement can be best accomplished if at least three image-guided core biopsies be obtained per sample, each of these cores be examined for tumor cellularity and that at least some of them be freshly snap frozen in liquid nitrogen.

Glycoproteomic comparison of clinical triple-negative and luminal breast tumors.

Triple-negative (TN) breast cancer accounts for ∼ 15% of breast cancers and is characterized by a high likelihood of relapse and a lack of targeted therapies. In contrast, luminal-type tumors that express the estrogen and progesterone receptors (ER+/PR+) and lack expression of human epidermal growth factor receptor 2 (Her2-) are treated with targeted hormonal therapy and carry a better prognosis. To identify potential targets for the development of future therapeutics aimed specifically at TN breast cancers, we have used a hydrazide-based glycoproteomic workflow to compare protein expression in clinical tumors from nine TN (Her2-/ER-/PR-) and nine luminal (Her2-/ER+/PR+) patients. Using a label-free LC-MS based approach, we identified and quantified 2264 proteins. Of these, 90 proteins were more highly expressed and 86 proteins were underexpressed in the TN tumors relative to the luminal tumors. The expression level of four of these potential targets was verified in the original set of tumors by Western blot and correlated well with our mass-spectrometry-based quantification. Furthermore, 30% of the proteins differentially expressed between luminal and TN tumors were validated in a larger cohort of 406 TN and 469 luminal tumors through corresponding differences in their mRNA expression in publically available microarray data. A group of 29 of these differentially expressed proteins was shown to correctly classify 88% of TN and luminal tumors using microarray data of their associated mRNA levels. Interestingly, even within a group of TN breast cancer patients, the expression levels of these same mRNAs were able to significantly predict patient survival, suggesting that these proteins play a role in the aggressiveness seen in TN tumors. This study provides a comprehensive list of potential targets for the development of diagnostic and therapeutic agents specifically aimed at treating TN breast cancer and demonstrates the utility of using publicly available microarray data to further prioritize potential targets.

Next-generation biobanking of metastases to enable multidimensional molecular profiling in personalized medicine.

Great advances in analytical technology coupled with accelerated new drug development and growing understanding of biological challenges, such as tumor heterogeneity, have required a change in the focus for biobanking. Most current banks contain samples of primary tumors, but linking molecular signatures to therapeutic questions requires serial biopsies in the setting of metastatic disease, next-generation of biobanking. Furthermore, an integration of multidimensional analysis of various molecular components, that is, RNA, DNA, methylome, microRNAome and post-translational modifications of the proteome, is necessary for a comprehensive view of a tumor's biology. While data using such biopsies are now regularly presented, the preanalytical variables in tissue procurement and processing in multicenter studies are seldom detailed and therefore are difficult to duplicate or standardize across sites and across studies. In the context of a biopsy-driven clinical trial, we generated a detailed protocol that includes morphological evaluation and isolation of high-quality nucleic acids from small needle core biopsies obtained from liver metastases. The protocol supports stable shipping of samples to a central laboratory, where biopsies are subsequently embedded in support media. Designated pathologists must evaluate all biopsies for tumor content and macrodissection can be performed if necessary to meet our criteria of >60% neoplastic cells and <20% necrosis for genomic isolation. We validated our protocol in 40 patients who participated in a biopsy-driven study of therapeutic resistance in metastatic colorectal cancer. To ensure that our protocol was compatible with multiplex discovery platforms and that no component of the processing interfered with downstream enzymatic reactions, we performed array comparative genomic hybridization, methylation profiling, microRNA profiling, splicing variant analysis and gene expression profiling using genomic material isolated from liver biopsy cores. Our standard operating procedures for next-generation biobanking can be applied widely in multiple settings, including multicentered and international biopsy-driven trials.

A micro-flow, high-pH, reversed-phase peptide fractionation and collection system for targeted and in-depth proteomics of low-abundance proteins in limiting samples.

We present a method and a simple system for high-pH RP-LC peptide fractionation of small sample amounts (30-60 µg), at micro-flow rates with micro-liter fraction collection using ammonium bicarbonate as an optimized buffer for system stability and robustness. The method is applicable to targeted mass spectrometry approaches and to in-depth proteomic studies where the amount of sample is limited. Using targeted proteomics with peptide standards, we present the method's analytical parameters, and potential in increasing the detection of low-abundance proteins that are difficult to quantify with direct targeted or global LC-MS analyses. This fractionation system increased peptide signals by up to 18-fold, while maintaining high quantitative precision, with high fractionation reproducibility across varied sample sets. In real applications, it increased the detection of targeted endogenous peptides by two-fold in a 25 cell-cycle-control protein panel, and in-depth MS analyses of nuclear extracts, it allowed the detection of up to 8,896 proteins with 138,417 peptides in 24-concatenated fractions compared to 3,344 proteins with 23,093 peptides without fractionation. In a relevant biological problem of CDK4/6-inhibitors and breast cancer, the method reproduced known information and revealed novel insights, highlighting that it can be successfully applied in studies involving low-abundance proteins and limited samples. •Tested nine high-pH buffer/solvent systems to obtain a robust, effective, and reproducible micro-flow fractionation method which was devoid of commonly encountered LC clogging/pressure issues after months of use.•Peptide enrichment method to improve detection and quantitation of low-abundance proteins in targeted and in-depth proteomic studies.•Can be applied to diverse protein samples where the available amount is limited.

The Mutational Spectrum of Pre- and Post-Neoadjuvant Chemotherapy Triple-Negative Breast Cancers.

The response of triple-negative breast cancer (TNBC) patients to pre-operative (neoadjuvant chemotherapy) is a critical factor of their outcome. To determine the effects of chemotherapy on the tumor genome and to identify mutations associated with chemoresistance and sensitivity, we performed whole exome sequencing on pre/post-chemotherapy tumors and matched lymphocytes from 26 patients. We observed great inter-tumoral heterogeneity with no gene mutated recurrently in more than four tumors besides TP53. Although the degree of response to chemotherapy in residual tumors was associated with more subclonal changes during chemotherapy, there was minimal evolution between pre/post-tumors. Indeed, gene sets enriched for mutations in pre- and post-chemotherapy tumors were very similar and reflected genes involved in the biological process of neurogenesis. Somatically mutated genes present in chemosensitive tumors included COL1A2, PRMD15, APOBEC3B, PALB2 and histone protein encoding genes, while BRCA1, ATR, ARID1A, XRCC3 and genes encoding for tubulin-associated proteins were present in the chemoresistant tumors. We also found that the mutational spectrum of post-chemotherapy tumors was more reflective of matching metastatic tumor biopsies than pre-chemotherapy samples. These findings support a portrait of modest ongoing genomic instability with respect to single-nucleotide variants induced by or selected for by chemotherapy in TNBCs.

Healthcare policy by other means: Cancer clinical research as "oncopolicy".

Social studies of biomedicine often focus on how exogenous policies shape the medical domain. While policy agendas no doubt affect complex biomedical projects, in the present paper we analyze a different dynamic, namely how oncologists enact policy as part of several flagship precision oncology endeavors. Empirically, the article focuses on the U.S. TAPUR trial, the Dutch DRUP trial, and the Canadian CAPTUR trial, which have recently been joined by similar Scandinavian studies. Taken together, these trials represent innovative forms of clinical research that, beyond their varying experimental nature, have been designed to transform the evidential processes to provide access to biomarker-driven treatments. Along with gathering evidence on effectiveness of off-label targeted therapies, their explicit goals include the recentering of a major professional organization around research, and the reframing of healthcare as a learning system seamlessly connecting epistemic, organizational, and economic issues. Accordingly, we analyze the design and implementation of these trials as a form of (onco)policy by other means.

HSP90 inhibitors induce GPNMB cell-surface expression by modulating lysosomal positioning and sensitize breast cancer cells to glembatumumab vedotin.

Transmembrane glycoprotein NMB (GPNMB) is a prognostic marker of poor outcome in patients with triple-negative breast cancer (TNBC). Glembatumumab Vedotin, an antibody drug conjugate targeting GPNMB, exhibits variable efficacy against GPNMB-positive metastatic TNBC as a single agent. We show that GPNMB levels increase in response to standard-of-care and experimental therapies for multiple breast cancer subtypes. While these therapeutic stressors induce GPNMB expression through differential engagement of the MiTF family of transcription factors, not all are capable of increasing GPNMB cell-surface localization required for Glembatumumab Vedotin inhibition. Using a FACS-based genetic screen, we discovered that suppression of heat shock protein 90 (HSP90) concomitantly increases GPNMB expression and cell-surface localization. Mechanistically, HSP90 inhibition resulted in lysosomal dispersion towards the cell periphery and fusion with the plasma membrane, which delivers GPNMB to the cell surface. Finally, treatment with HSP90 inhibitors sensitizes breast cancers to Glembatumumab Vedotin in vivo, suggesting that combination of HSP90 inhibitors and Glembatumumab Vedotin may be a viable treatment strategy for patients with metastatic TNBC.

CXCR4 peptide antagonist inhibits primary breast tumor growth, metastasis and enhances the efficacy of anti-VEGF treatment or docetaxel in a transgenic mouse model.

CXCR4 is a chemokine receptor implicated in the homing of cancer cells to target metastatic organs, which overexpress its ligand, stromal cell-derived factor (SDF)-1. To determine the efficacy of targeting CXCR4 on primary tumor growth and metastasis, we used a peptide inhibitor of CXCR4, CTCE-9908, that was administered in a clinically relevant approach using a transgenic breast cancer mouse model. We first performed a dosing experiment of CTCE-9908 in the PyMT mouse model, testing 25, 50 and 100 mg/kg versus the scrambled peptide in groups of 8-16 mice. We then combined CTCE-9908 with docetaxel or DC101 (an anti-VEGFR2 monoclonal antibody). We found that increasing doses of CTCE-9908 alone slowed the rate of tumor growth, with a 45% inhibition of primary tumor growth at 3.5 weeks of treatment with 50 mg/kg of CTCE-9908 (p = 0.005). Expression levels of VEGF were also found to be reduced by 42% with CTCE-9908 (p = 0.01). In combination with docetaxel, CTCE-9908 administration decreased tumor volume by 38% (p = 0.02), an effect that was greater than that observed with docetaxel alone. In combination with DC101, CTCE-9908 also demonstrated an enhanced effect compared to DC101 alone, with a 37% decrease in primary tumor volume (p = 0.01) and a 75% reduction in distant metastasis (p = 0.009). In combination with docetaxel or an anti-angiogenic agent, the anti-tumor and anti-metastatic effects of CTCE-9908 were markedly enhanced, suggesting potentially new effective combinatorial therapeutic strategies in the treatment of breast cancer, which include targeting the SDF-1/CXCR4 ligand/receptor pair.

Mesenchymal stromal cells ameliorate experimental autoimmune encephalomyelitis by inhibiting CD4 Th17 T cells in a CC chemokine ligand 2-dependent manner.

The administration of ex vivo culture-expanded mesenchymal stromal cells (MSCs) has been shown to reverse symptomatic neuroinflammation observed in experimental autoimmune encephalomyelitis (EAE). The mechanism by which this therapeutic effect occurs remains unknown. In an effort to decipher MSC mode of action, we found that MSC conditioned medium inhibits EAE-derived CD4 T cell activation by suppressing STAT3 phosphorylation via MSC-derived CCL2. Further analysis demonstrates that the effect is dependent on MSC-driven matrix metalloproteinase proteolytic processing of CCL2 to an antagonistic derivative. We also show that antagonistic CCL2 suppresses phosphorylation of AKT and leads to a reciprocal increased phosphorylation of ERK associated with an up-regulation of B7.H1 in CD4 T cells derived from EAE mice. CD4 T cell infiltration of the spinal cord of MSC-treated group was robustly decreased along with reduced plasma levels of IL-17 and TNF-alpha levels and in vitro from restimulated splenocytes. The key role of MSC-derived CCL2 was confirmed by the observed loss of function of CCL2(-/-) MSCs in EAE mice. In summary, this is the first report of MSCs modulating EAE biology via the paracrine conversion of CCL2 from agonist to antagonist of CD4 Th17 cell function.

Early, On-Treatment Levels and Dynamic Changes of Genomic Instability in Circulating Tumor DNA Predict Response to Treatment and Outcome in Metastatic Breast Cancer Patients.

BACKGROUND: Circulating tumor DNA (ctDNA) offers high sensitivity and specificity in metastatic cancer. However, many ctDNA assays rely on specific mutations in recurrent genes or require the sequencing of tumor tissue, difficult to do in a metastatic disease. The purpose of this study was to define the predictive and prognostic values of the whole-genome sequencing (WGS) of ctDNA in metastatic breast cancer (MBC). METHODS: Plasma from 25 patients with MBC were taken at the baseline, prior to treatment (T0), one week (T1) and two weeks (T2) after treatment initiation and subjected to low-pass WGS. DNA copy number changes were used to calculate a Genomic Instability Number (GIN). A minimum predefined GIN value of 170 indicated detectable ctDNA. GIN values were correlated with the treatment response at three and six months by Response Evaluation Criteria in Solid Tumours assessed by imaging (RECIST) criteria and with overall survival (OS). RESULTS: GIN values were detectable (>170) in 64% of patients at the baseline and were significantly prognostic (41 vs. 18 months OS for nondetectable vs. detectable GIN). Detectable GIN values at T1 and T2 were significantly associated with poor OS. Declines in GIN at T1 and T2 of > 50% compared to the baseline were associated with three-month response and, in the case of T1, with OS. On the other hand, a rise in GIN at T2 was associated with a poor response at three months. CONCLUSIONS: Very early measurements using WGS of cell-free DNA (cfDNA) from the plasma of MBC patients provided a tumor biopsy-free approach to ctDNA measurement that was both predictive of the early tumor response at three months and prognostic.

STAT1 potentiates oxidative stress revealing a targetable vulnerability that increases phenformin efficacy in breast cancer.

Bioenergetic perturbations driving neoplastic growth increase the production of reactive oxygen species (ROS), requiring a compensatory increase in ROS scavengers to limit oxidative stress. Intervention strategies that simultaneously induce energetic and oxidative stress therefore have therapeutic potential. Phenformin is a mitochondrial complex I inhibitor that induces bioenergetic stress. We now demonstrate that inflammatory mediators, including IFNγ and polyIC, potentiate the cytotoxicity of phenformin by inducing a parallel increase in oxidative stress through STAT1-dependent mechanisms. Indeed, STAT1 signaling downregulates NQO1, a key ROS scavenger, in many breast cancer models. Moreover, genetic ablation or pharmacological inhibition of NQO1 using ß-lapachone (an NQO1 bioactivatable drug) increases oxidative stress to selectively sensitize breast cancer models, including patient derived xenografts of HER2+ and triple negative disease, to the tumoricidal effects of phenformin. We provide evidence that therapies targeting ROS scavengers increase the anti-neoplastic efficacy of mitochondrial complex I inhibitors in breast cancer.

Both t-Darpp and DARPP-32 can cause resistance to trastuzumab in breast cancer cells and are frequently expressed in primary breast cancers.

The clinical use of trastuzumab (Herceptin), a humanized antibody against the HER2 growth factor receptor, has improved survival in patients with breast tumors with ERBB2 amplification and/or over-expression. However, most patients with advanced ERBB2 amplified breast cancers whose tumors initially respond to trastuzumab develop resistance to the drug, leading to tumor progression. To identify factors responsible for acquired resistance to trastuzumab, gene expression profiling was performed on subclones of an ERBB2 amplified breast cancer cell line, BT474, which had acquired resistance to trastuzumab. The most overexpressed gene in these subclones was PPP1R1B, encoding the DARPP-32 phosphatase inhibitor. Western analysis revealed that only the truncated isoform of the DARPP-32 protein, t-Darpp, was overexpressed in the trastuzumab resistant cells. Using gene silencing experiments, we confirmed that t-Darpp over-expression was required for trastuzumab resistance in these cells. Furthermore, transfecting t-Darpp in parental BT-474 cells conferred resistance to trastuzumab, suggesting that t-Darpp expression was sufficient for trastuzumab resistance. We also found that t-Darpp over-expression was associated with Akt activation and that the T75 residue in t-Darpp was required for both Akt activation and trastuzumab resistance. Finally, we found that full-length DARPP-32 and t-Darpp are expressed in a majority of primary breast tumors. Over-expression of full-length DARPP-32 can also confer resistance to trastuzumab and, moreover, is associated with a poor prognostic value in breast cancers. Thus, t-Darpp and DARPP-32 expression are novel prognostic and predictive biomarkers in breast cancer.