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Supplemental Figure 1-2. Supplemental Figure 1: Pharmacology of POL5551; Supplemental Figure 2: Correlation of CXCR4 expression in primary tumor with survival in patients

Supplemental Figure 5. Eribulin dose ranging study.

PDF file - 9.8MB, Temporal and Spatial cooperation of Snail1 and Twist1 during Epithelial-Mesenchymal Transition predicts for human breast cancer recurrence.

Supplemental Figure 3. Gaussia luciferase complementation imaging model and scratch migration assay with AMD3100.

Supplemental Figure 4. MDA-MB-231 primary tumor weight and liver metastases data.

Supplemental Figure 6. Mobilization of leukocytes with POL5551.

The SDF-1 receptor CXCR4 has been associated with early metastasis and poorer prognosis in breast cancers, especially the most aggressive triple-negative subtype. In line with previous reports, we found that tumoral CXCR4 expression in patients with locally advanced breast cancer was associated with increased metastases and rapid tumor progression. Moreover, high CXCR4 expression identified a group of bone marrow–disseminated tumor cells (DTC)-negative patients at high risk for metastasis and death. The protein epitope mimetic (PEM) POL5551, a novel CXCR4 antagonist, inhibited binding of SDF-1 to CXCR4, had no direct effects on tumor cell viability, but reduced migration of breast cancer cells in vitro. In two orthotopic models of triple-negative breast cancer, POL5551 had little inhibitory effect on primary tumor growth, but significantly reduced distant metastasis. When combined with eribulin, a chemotherapeutic microtubule inhibitor, POL5551 additively reduced metastasis and prolonged survival in mice after resection of the primary tumor compared with single-agent eribulin. Hypothesizing that POL5551 may mobilize tumor cells from their microenvironment and sensitize them to chemotherapy, we used a “chemotherapy framing” dosing strategy. When administered shortly before and after eribulin treatment, three doses of POL5551 with eribulin reduced bone and liver tumor burden more effectively than chemotherapy alone. These data suggest that sequenced administration of CXCR4 antagonists with cytotoxic chemotherapy synergize to reduce distant metastases. Mol Cancer Ther; 14(11); 2473–85. ©2015 AACR.

Supplemental Table 1-3. Pharmacology of POL5551 and patient characteristics at baseline

Supplemental Figure 7. Effect of POL5551 combined with eribulin on hematopoietic cells

Supplementary Table 1 from Accuracy of Perceived Risk of Recurrence Among Patients With Early-Stage Breast Cancer

Purpose: Micrometastatic cells detected in the bone marrow have prognostic significance in breast cancer. These cells are heterogeneous and likely do not exhibit uniform biological behavior. To understand the molecular diversity of disseminated cancer cells that reside in bone marrow, we enriched this cell population and did global gene expression profiling in the context of a prospective clinical trial involving women with clinical stage II/III breast cancer undergoing neoadjuvant chemotherapy.Experimental Design: Enrichment of TACSTD1 (EpCAM)–expressing cells from bone marrow of breast cancer patients was achieved using immunomagnetic beads. Gene expression profiles were compared between enriched cell populations and whole bone marrow from 5 normal volunteers and 23 breast cancer patients after neoadjuvant chemotherapy treatment. Enriched cells from bone marrow samples of breast cancer patients before treatment or at 1 year follow-up were also analyzed (total of 87 data sets). The expression of transcripts specifically detected in enriched cell populations from breast cancer patients was correlated with 1-year clinical outcome using quantitative reverse transcription-PCR in an independent cohort of bone marrow samples.Results: Analysis of EpCAM-enriched bone marrow cells revealed specific expression of a subgroup of transcripts, including the metastasis regulator, TWIST1. Most transcripts identified, including TWIST1, were not expressed in enriched populations of bone marrow from normal volunteers, suggesting that this expression profile reflects a signature of breast cancer bone marrow micrometastases that persist after chemotherapy. In an independent set of bone marrow samples obtained before any treatment, TWIST1 expression correlated with early disease relapse.Conclusions: Disseminated breast cancer cells present in bone marrow after chemotherapy possess unique transcriptional signatures. Genes whose expression is overrepresented in these cell populations, such as TWIST1, may prove to be excellent markers of early distant relapse in breast cancer patients.

Supplementary Table S2 from Isolation and Molecular Profiling of Bone Marrow Micrometastases Identifies TWIST1 as a Marker of Early Tumor Relapse in Breast Cancer Patients

Crucial transitions in cancer-including tumor initiation, local expansion, metastasis, and therapeutic resistance-involve complex interactions between cells within the dynamic tumor ecosystem. Transformative single-cell genomics technologies and spatial multiplex in situ methods now provide an opportunity to interrogate this complexity at unprecedented resolution. The Human Tumor Atlas Network (HTAN), part of the National Cancer Institute (NCI) Cancer Moonshot Initiative, will establish a clinical, experimental, computational, and organizational framework to generate informative and accessible three-dimensional atlases of cancer transitions for a diverse set of tumor types. This effort complements both ongoing efforts to map healthy organs and previous large-scale cancer genomics approaches focused on bulk sequencing at a single point in time. Generating single-cell, multiparametric, longitudinal atlases and integrating them with clinical outcomes should help identify novel predictive biomarkers and features as well as therapeutically relevant cell types, cell states, and cellular interactions across transitions. The resulting tumor atlases should have a profound impact on our understanding of cancer biology and have the potential to improve cancer detection, prevention, and therapeutic discovery for better precision-medicine treatments of cancer patients and those at risk for cancer.

Intratumoral heterogeneity, which is manifested in almost all of the hallmarks of cancer, including the significantly altered metabolic profiles of cancer cells, represents a challenge to effective cancer therapy. High-throughput measurements of the metabolism of individual cancer cells would allow direct visualization and quantification of intratumoral metabolic heterogeneity, yet the throughputs of current measurement techniques are limited to about 120 cells per hour. Here, we show that single-cell photoacoustic microscopy can reach throughputs of approximately 12,000 cells per hour by trapping single cells with blood in an oxygen-diffusion-limited high-density microwell array and by using photoacoustic imaging to measure the haemoglobin oxygen change (that is, the oxygen consumption rate) in the microwells. We demonstrate the capability of this label-free technique by performing high-throughput single-cell oxygen-consumption-rate measurements of cultured cells and by imaging intratumoral metabolic heterogeneity in specimens from patients with breast cancer. High-throughput single-cell photoacoustic microscopy of oxygen consumption rates should enable the faster characterization of intratumoral metabolic heterogeneity.

This first study in humans was designed to evaluate the safety and dosimetry of a cellular proliferative marker, N-(4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)butyl)-2-(2-18F-fluoroethoxy)-5-methylbenzamide (18F-ISO-1), and evaluate the feasibility of imaging tumor proliferation by PET in patients with newly diagnosed malignant neoplasms. Methods: Patients with biopsy-proven lymphoma, breast cancer, or head and neck cancer underwent 18F-ISO-1 PET. Tumor 18F-ISO-1 uptake was assessed semiquantitatively by maximum standardized uptake value, ratios of tumor to normal tissue and tumor to muscle, and relative distribution volume ratio. The PET results were correlated with tumor Ki-67 and mitotic index, from in vitro assays of the tumor tissue. The biodistribution of 18F-ISO-1 and human dosimetry were evaluated. Results: Thirty patients with primary breast cancer (n = 13), head and neck cancer (n = 10), and lymphoma (n = 7) were evaluated. In the entire group, tumor maximum standardized uptake value and tumor-to-muscle ratio correlated significantly with Ki-67 (τ = 0.27, P = 0.04, and τ = 0.38, P = 0.003, respectively), but no significant correlation was observed between Ki-67 and tumor–to–normal-tissue ratio (τ = 0.07, P = 0.56) or distribution volume ratio (τ = 0.26, P = 0.14). On the basis of whole-body PET data, the gallbladder is the dose-limiting organ, with an average radiation dose of 0.091 mGy/MBq. The whole-body and effective doses were 0.012 mGy/MBq and 0.016 mSv/MBq, respectively. No adverse effects of 18F-ISO-1 were encountered. Conclusion: The presence of a significant correlation between 18F-ISO-1 and Ki-67 makes this agent promising for evaluation of the proliferative status of solid tumors. The relatively small absorbed doses to normal organs allow for the safe administration of up to 550 MBq, which is sufficient for PET imaging in clinical trials.

The goal of the current study is to identify predictors responsible for mortality disparities between young (or =40 years) and older (40 years) women with breast cancer.From 1998 to 2006, 344 patientsor =40 years were treated for breast cancer. Cox regression models calculated adjusted hazard ratios (aHR) and 95% confidence intervals (CI) to determine differences in breast cancer mortality in womenor =40 years versus40 years (n = 3,252), controlling for potential confounders in univariate tests.From 1998 to 2006, 3,596 patients were treated for breast cancer; 9.6% wereor =40 years and 90.4% were40 years. Young women were more likely to be African-American, with a family history of breast cancer, diagnosed at advanced stage, and treated by mastectomy (P0.05). Tumors in young women were more likely to be bilateral, T2/T3, grade III, ER/PR negative, and lymph-node positive (P0.01). Overall, young women (or =40 years) with breast cancer were more likely to die compared with older women (40 years) (aHR 1.52, CI 1.37-1.74).Young women (or =40 years) with breast cancer are diagnosed at a more advanced stage and have tumors with poor prognostic features. Young women (or =40 years) are 52% more likely to die from breast cancer compared to older women (40 years).

Most cancer deaths are a consequence of resistance to conventional chemotherapy and radiation therapy. This may be attributable to unique phenotypic characteristics of solid tumors. We have exploited two well-described characteristics of solid tumors commonly associated with treatment failure, high glucose use and hypoxia, to design a unique therapy based on the selective accumulation of two cytotoxic compounds, 2-deoxyglucose (2-DG) and copper(II)diacetyl-bis(N(4)-methylthiosemicarbazone) ((64)Cu-ATSM). (64)Cu-ATSM localizes to hypoxic regions of tumors and has been used for administering a high local dose of radiation therapy after uptake by cells. 2-DG, a glucose analog, selectively accumulates in cancer cells and interferes with energy metabolism, resulting in cancer cell death. 2-DG has been shown to potentiate the cytotoxic effect of ionizing radiation and certain chemotherapeutic agents. We have tested the effect of 2-DG on tumor response when combined with (64)Cu-ATSM in a mouse breast tumor model using the highly aggressive mouse mammary carcinoma cell line EMT-6. 2-DG administered up to 2 mg/g of body weight daily resulted in no weight loss or systemic symptoms. EMT-6 mammary tumors had similar uptake of [(18)F]fluoro-2-deoxyglucose before and after 2 weeks of 2-DG treatment as determined by microPET imaging, indicating that resistance to 2-DG uptake does not develop. Pretreatment of tumor-bearing mice with 2-DG resulted in increased uptake of (64)Cu-ATSM by tumors compared with nontreated mice. This effect was not observed with the nonhypoxia-specific agent copper(II)pyruvaldehyde-bis(N(4)-methylthiosemicarbazone. When 2-DG was combined with a single dose of (64)Cu-ATSM (2 mCi), tumor growth was inhibited approximately 60% compared with untreated mice, and animals survived approximately 50% longer than untreated mice or animals treated with each agent alone (32 versus 20 days). The maximum effect on tumor growth and survival was observed when 2-DG was administered daily for the lifetime of the mouse. Our results indicate that 2-DG potentiates the effect of (64)Cu-ATSM on tumoricidal activity and animal survival. We hypothesize that 2-DG alters the metabolic state of the cell, leading to increased uptake of (64)Cu-ATSM by the tumor. This would result in a higher local dose of radiotherapy. The continued presence of 2-DG would then prevent the repair of damaged cells, leading to inhibition of tumor growth. Our data indicate that the strategy of combining tumor-specific cytotoxic agents that function by differing mechanisms can result in an effective, selective, tumor-specific cell death with minimal effect on the host.

This study examined whether disparities in mammography use between women of differing socioeconomic status (SES; income and education) and varying access to medical care (healthcare insurance and routine medical check-up) remained over time despite overall increased breast cancer screening.Analysis of changes over time were made using data from the 1992, 1996, and 2000 Behavioral Risk Factor Surveillance System data from 53,846 women 50-69 years of age. Women who reported that they never had a mammogram were compared with those who ever had a mammogram. Multivariate logistic regression was used to determine whether and to what extent disparities between subgroups of women changed over time.The percentage of women 50-69 years of age who had never had a mammogram declined 65% from 22.1% in 1992 to 7.7% in 2000. Racial and ethnic differences in mammography prevalence disappeared over time. However, disparities among women of differing SES and among those with varying access to medical care remained based on multivariate analysis.Despite a substantial reduction in the proportion of women who had never had a mammogram among women 50-69 years of age from 1992 to 2000, disparities in use of mammography among the various population subgroups persisted.

Exposure to ionizing radiation is believed to cause cell injury via the production of free radicals that are thought to induce oxidative damage. It has been proposed that exposure to agents that enhance oxidative stress-induced injury by disrupting thiol metabolism may sensitize cells to the cytotoxic effects of ionizing radiation. Recently, it has been shown that glucose deprivation selectively induces cell injury in transformed human cells via metabolic oxidative stress (J. Biol. Chem., 273: 5294-5299; Ann. N.Y. Acad. Sci., 899: 349-362), resulting in profound disruptions in thiol metabolism. Because 2-deoxy-D-glucose (2DG) is a potent inhibitor of glucose metabolism thought to mimic glucose deprivation in vivo, the hypothesis that exposure to 2DG might be capable of inducing radiosensitization in transformed cells via perturbations in thiol metabolism was tested. When HeLa cells were exposed to 2DG (4-10 mM) for 4-72 h, cell survival decreased (20-90%) in a dose- and time-dependent fashion. When HeLa cells were treated with 6 mM 2DG for 16 h before ionizing radiation exposure, radiosensitization was observed with a sensitizer enhancement ratio of 1.4 at 10% isosurvival. Treatment with 2DG was also found to cause decreases in intracellular total glutathione content (50%). Simultaneous treatment with the thiol antioxidant N-acetylcysteine (NAC; 30 mM) protected HeLa cells against the cytotoxicity and radiosensitizing effects of 2DG, without altering radiosensitivity in the absence of 2DG. Furthermore, treatment with NAC partially reversed the 2DG-induced decreases in total glutathione content, as well as augmented intracellular cysteine content. Finally, the cytotoxicity and radiosensitizing effects of 2DG were more pronounced in v-Fos-transformed versus nontransformed immortalized rat cells, and this radiosensitization was also inhibited by treatment with NAC. These results support the hypothesis that exposure to 2DG causes cytotoxicity and radiosensitization via a mechanism involving perturbations in thiol metabolism and allows for the speculation that these effects may be more pronounced in transformed versus normal cells.

Hemin (ferriprotoporphyrin IX-chloride) can mediate the covalent cross-linking and degradation of yeast glutathione reductase. This reaction requires both NADPH and oxygen suggesting the involvement of a reduced oxygen species in the cross-linking and degradation process. During the course of the reaction the enzymatic activity of glutathione reductase is rapidly destroyed. Implications of these findings for a regulatory role of hemin in cell biology are discussed.

Low levels of dexamethasone and related glucocorticoid hormones suppress the expression of globin genes during the DMSO-induced differentiation of Friend leukemia cells. In this response, the glucocorticoids appear to act at both the transcriptional and post-transcriptional levels in that 10(-8) M dexamethasone prevents the accumulation of both globin mRNA and globin protein, whereas 10(-9) M dexamethasone allows the accumulation of normal levels of hybridizable globin mRNA but prevents the accumulation of globin protein. This suppressive action of dexamethasone is more effective with DMSO as the inducer of globin gene expression than with hemin as the inducer. In contrast to the situation with glucocorticoids, certain sex steroids (etiocholanolone, testosterone and estradiol) facilitate the expression of globin genes in DMSO-treated Friend leukemia cells. The modulation of globin gene expression by steroid hormones is achieved in DMSO-treated cells without altering the growth and morphological changes which characteristically attend the differentiation of these cells.