Search

Supplementary Table 1 and Figure 4 from Conservation of Genetic Alterations in Recurrent Melanoma Supports the Melanoma Stem Cell Hypothesis

Supplementary Data 1 from Conservation of Genetic Alterations in Recurrent Melanoma Supports the Melanoma Stem Cell Hypothesis

It is generally accepted that human cancers derive from a mutated single cell. However, the genetic steps characterizing various stages of progression remain unclear. Studying a unique case of metastatic melanoma, we observed that cell lines derived from metachronous metastases arising over a decade retained a central core of genetic stability in spite of divergent phenotypes. In the present study, we expanded our previous observations comparing these autologous cell lines of clonal derivation with allogeneic ones and correlated array comparative genomic hybridization (aCGH) with gene expression profiling to determine their relative contribution to the dynamics of disease progression. aCGH and gene expression profiling were performed on autologous cell lines and allogeneic melanoma cell lines originating from other patients. A striking correlation existed between total extent of genetic imbalances, global transcriptional patterns, and cellular phenotypes. They did not follow a strict temporal progression but stemmed independently at various time points from a central core of genetic stability best explained according to the cancer stem cell hypothesis. Although their contribution was intertwined, genomic imbalances detectable by aCGH contributed only 25% of the transcriptional traits determining autologous tumor distinctiveness. Our study provides important insights about the dynamics of cancer progression and supports the development of targeted anticancer therapies aimed against stable genetic factors that are maintained throughout the end stage of disease. [Cancer Res 2008;68(1):122–31]

There is an increasing interest in complementing conventional histopathologic evaluation with molecular tools that could increase the sensitivity and specificity of cancer staging for diagnostic and prognostic purposes. This study strove to identify cancer-specific markers for the molecular detection of a broad range of cancer types. We used 373 archival samples inclusive of normal tissues of various lineages and benign or malignant tumors (predominantly colon, melanoma, ovarian, and esophageal cancers). All samples were processed identically and cohybridized with an identical reference RNA source to a custom-made cDNA array platform. The database was split into training (n = 201) and comparable prediction (n = 172) sets. Leave-one-out cross-validation and gene pairing analysis identified putative cancer biomarkers overexpressed by malignant lesions independent of tissue of derivation. In particular, seven gene pairs were identified with high predictive power (87%) in segregating malignant from benign lesions. Receiver operator characteristic curves based on the same genes could segregate malignant from benign tissues with 94% accuracy. The relevance of this study rests on the identification of a restricted number of biomarkers ubiquitously expressed by cancers of distinct histology. This has not been done before. These biomarkers could be used broadly to increase the sensitivity and accuracy of cancer staging and early detection of locoregional or systemic recurrence. Their selective expression by cancerous compared with paired normal tissues suggests an association with the oncogenic process resulting in stable expression during disease progression when the presently used differentiation markers are unreliable. (Cancer Res 2006; 66(6): 2953-61)

Supplementary Table from Common Cancer Biomarkers

Pathologists rely on microscopy to diagnose disease states in tissues and organs. They utilize both high-resolution, high-magnification images to interpret the staining and morphology of individual cells, as well as low-magnification overviews to give context and location to these cells. Intravital imaging is a powerful technique for studying cells and tissues in their native, live environment and can yield sub-cellular resolution images similar to those used by pathologists. However, technical limitations prevent the straightforward acquisition of low-magnification images during intravital imaging, and they are hence not typically captured. The serial acquisition, mosaicking, and stitching together of many high-resolution, high-magnification fields of view is a technique that overcomes these limitations in fixed and ex vivo tissues. The technique however, has not to date been widely applied to intravital imaging as movements caused by the living animal induce image distortions that are difficult to compensate for computationally. To address this, we have developed techniques for the stabilization of numerous tissues, including extremely compliant tissues, that have traditionally been extremely difficult to image. We present a novel combination of these stabilization techniques with mosaicked and stitched intravital imaging, resulting in a process we call Large-Volume High-Resolution Intravital Imaging (LVHR-IVI). The techniques we present are validated and make large volume intravital imaging accessible to any lab with a multiphoton microscope.

Stable, high-resolution intravital imaging of the lung has become possible through the utilization of vacuum-stabilized imaging windows. However, this technique is extremely invasive and limited to only hours in duration. Here we describe a minimally invasive, permanently implantable window for high-resolution intravital imaging of the murine lung that allows the mouse to survive surgery, recover from anesthesia, and breathe independently. Compared to vacuum-stabilized windows, this window produces the same high-quality images without vacuum-induced artifacts; it is also less invasive, which allows imaging of the same lung tissue over a period of weeks. We further adapt the technique of microcartography for reliable relocalization of the same cells longitudinally. Using commonly employed experimental, as well as more clinically relevant, spontaneous metastasis models, we visualize all stages of metastatic seeding, including: tumor cell arrival; extravasation; growth and progression to micrometastases; as well as tumor microenvironment of metastasis function, the hallmark of hematogenous dissemination of tumor cells.

It is generally accepted that human cancers derive from a mutated single cell. However, the genetic steps characterizing various stages of progression remain unclear. Studying a unique case of metastatic melanoma, we observed that cell lines derived from metachronous metastases arising over a decade retained a central core of genetic stability in spite of divergent phenotypes. In the present study, we expanded our previous observations comparing these autologous cell lines of clonal derivation with allogeneic ones and correlated array comparative genomic hybridization (aCGH) with gene expression profiling to determine their relative contribution to the dynamics of disease progression. aCGH and gene expression profiling were performed on autologous cell lines and allogeneic melanoma cell lines originating from other patients. A striking correlation existed between total extent of genetic imbalances, global transcriptional patterns, and cellular phenotypes. They did not follow a strict temporal progression but stemmed independently at various time points from a central core of genetic stability best explained according to the cancer stem cell hypothesis. Although their contribution was intertwined, genomic imbalances detectable by aCGH contributed only 25% of the transcriptional traits determining autologous tumor distinctiveness. Our study provides important insights about the dynamics of cancer progression and supports the development of targeted anticancer therapies aimed against stable genetic factors that are maintained throughout the end stage of disease. [Cancer Res 2008;68(1):122–31]

Metastatic disease is the major cause of cancer mortality and is responsible for over 1/2 million deaths each year in the U.S. alone. Of the three most common sites of metastasis observed clinically (bone, lung and liver), the most difficult to study using intravital microscopy is the lung; a delicate organ in perpetual motion. We have recently developed and validated an implantable, permanent, lung imaging window which allows high-resolution multiphoton imaging of the intact, breathing (without ventilation), murine lung over days to weeks of repeated imaging. This window does not use vacuum to immobilize the lung tissue thereby avoiding well know artifacts associated with vacuum lung windows. Using our new window, we have been able to visualize, with single cell resolution, the steps of metastasis in a clinically relevant, spontaneously-metastasizing murine breast cancer model. We present the first direct visualization of tumor cell arrival, extravasation, and progression to micro-metastases. Further, we have observed, for the first time, the activity of the tripartite structure called Tumor MicroEnvironment of Metastasis (TMEM) in metastatic lung lesions directly demonstrating the mechanism of dissemination from metastatic lung tumors. Using this approach to imaging live lung tissue serially, within a single animal, we are investigating the mechanisms underlying the fate of tumor cells arriving in the lung with the ultimate goal of directly identifying signals behind seeding and survival of metastatic tumor cells and their responses to interventions in real time. Citation Format: Sonia E. Voiculescu, Yarong Wang, Maja Oktay, John Condeelis, David Entenberg. A permanent lung imaging window reveals, for the first time, the steps of extravasation, seeding and growth of early metastatic dissemination [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3055. doi:10.1158/1538-7445.AM2017-3055

There is increasing consensus that cancer stem cells (CSC) play an important role during metastatic progression of breast cancer. Previous studies have shown that stem cells display a pro-invasive phenotype in breast cancer. However, the phenotypes specific to breast cancer stem cells have not been evaluated at single cell resolution in vivo. Here, we employ high-resolution intravital two-photon microscopy in both orthotopic xenograft tumors, and their metastases, formed from human breast cancer cell lines expressing a previously characterized SOX2/OCT4 transcription-based fluorescent stem cell biosensor (SORE6). Using this high resolution imaging technology we found that SORE6+ stem cells: (a) constitute a minority population of the primary mammary tumors, (b) move approximately ten times slower than non-stem breast cancer cells (0.1 vs 1.1 µm/min, respectively), (c) are migratory toward blood vessels and, (d) compared to non-stem cells, they are enriched for invasive cellular protrusions called invadopodia. This is important because we have shown that these phenotypes are specifically associated with the disseminating population of tumor cells in the primary tumor site and, in addition, invadopodia are required for transendothelial migration during intravasation. Stem cells also have a three-fold higher incidence of direct contact with macrophages compared to non-stem cells. In fact, stem cells were frequently seen to be part of the tripartite macrophage-tumor cell- endothelial cell complex called TMEM, which was previously shown to be the doorway for intravasation of tumor cells in primary mammary tumors and is validated as a prognostic of metastasis in human breast cancer patients. Furthermore, we followed breast cancer stem cell dissemination to the lung by using a novel lung window for high resolution imaging of the lung (WHRIL), which allows visualization of the same lung tissue in a single mouse, serially, over days to weeks. Using WHRIL, in combination with intravital multiphoton microscopy and the above biosensor, we visualized the arrival, extravasation and outgrowth of spontaneously arriving breast tumor cells in the lung. We report here for the first time, the kinetics and stemness state of the spontaneously metastasizing tumor cells and, the record at single cell resolution, of their fate and metastatic outgrowth in the lung over time. Citation Format: Ved P. Sharma, Sonia Voiculescu, Yarong Wang, George S. Karagiannis, Binwu Tang, Lalage Wakefield, David Entenberg, Sumanta Goswami, Maja Oktay, John Condeelis. A new SOX2/OCT4 stem cell biosensor reveals the mechanism of cancer stem cell dissemination in human breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 878. doi:10.1158/1538-7445.AM2017-878

Metastatic disease is the major cause of cancer mortality and is responsible for over 1/2 million deaths each year in the U.S. alone. Of the three most common sites of metastasis observed clinically (bone, lung and liver), the most difficult to study using intravital microscopy is the lung; a delicate organ in perpetual motion. Recent advances in intravital microscopy have enabled visualization of the live intact lung, but are limited in the duration over which they can be utilized (hours) and require major invasive surgeries. Both of these limitations reduce their usefulness in the study of single cell events in the lung and are susceptible to introducing artefacts. We report the development of a novel, minimally invasive surgical protocol for the implantation of a long-lasting lung imaging window. Once implanted, this window allows for the repeated, non-invasive visualization of living, breathing lung tissue over a period of two weeks without the need for ventilation, vacuum plates, or gated imaging equipment. Here we characterize and validate the use of this window in living mice. The window is well tolerated, does not cause infection or inflammation, and offers a 4mm view of the left lung. We utilize it to visualize, with single cell resolution, the progression of experimental metastasis from the arrival of a single cell to the formation of micro-metastases. The development of this novel optical window will allow visualization of, with single cell resolution, tumor microenvironments and metastatic progression within the lung over extended periods of time. This approach to imaging live lung tissue has the potential to reveal the mechanisms underlying many diseases affecting the lung as well as the means to directly evaluate responses to therapeutics in in real time and within a single animal. Citation Format: David Entenberg, Allison Harney, Sonia E. Voiculescu, Yarong Wang, Maja Oktay, John Condeelis. Characterization and validation of an implantable chronic lung imaging window. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4104.

Purpose: To describe the eicosanoid profile and differentially expressed eicosanoid and arachidonic acid pathway genes in tissues from patients with advanced epithelial ovarian cancer (EOC).Experimental Design: We first employed electrospray tandem mass spectrometry to determine tissue-specific concentrations of the eicosanoids prostaglandin E2 (PGE2), the hydroxyeicosatetraenoic acids (12-HETE and 5-HETE), and leukotriene (LTB4), selected for tumor growth potential, and two other bioactive lipids (15-HETE and 13-HODE) with tumor cell proliferation interference potential. The cellular location of eicosanoid activity was identified by immunofluorescence antibody costaining and confocal microscopy. Differential analysis of eicosanoid and arachidonic pathway genes was done using a previously validated cDNA microarray platform. Tissues used included EOC tumor, tumor-free malignant peritoneum (MP), and benign peritoneum (BP) from patients with benign pelvic disease.Results: (a) Eicosanoid products were detected in tumor, MP, and BP specimens. PGE2 levels were significantly elevated in tumors in an overall comparison with MP or BP (P < 0.001). Combined levels of PGE2, 12-HETE, 5-HETE, and LTB4 increased progressively from low to high concentrations in BP, MP, and tumors (P = 0.012). Neither 15-HETE nor 13-HODE showed a significant opposite trend toward levels found in BP. (b) Tissue specimens representing common EOC histotypes showed strong coexpressions of cyclooxygenases (COX-1) and prostaglandin E synthases (PGES-1) on tumor cells, whereas intratumoral or peritumoral MO/MA coexpressed COX-1 and COX-2 and PGES-1 and PGES-2, respectively. (c) cDNA microarray analysis of MP, BP, and tumor showed that a number of eicosanoid and arachidonic acid pathway genes were differentially expressed in MP and BP compared with tumor, except for CYP2J2, which was increased in tumors.Conclusions: Elevated levels of eicosanoid metabolites in tumors and differential expression of eicosanoid and arachidonic acid pathway genes in the peritoneum support the involvement of bioactive lipids in the inflammatory tumor environment of EOC.

Experimentally, interleukin-2 (IL-2) exerts complex immunological functions promoting the proliferation, survival and activation of T cells on one hand and inducing immune regulatory mechanisms on the other. This complexity results from a cross talk among immune cells which sways the effects of IL-2 according to the experimental or clinical condition tested. Recombinant IL-2 (rIL-2) stimulation of peripheral blood mononuclear cells (PBMC) from 47 donors of different genetic background induced generalized T cell activation and anti-apoptotic effects. Most effects were dependent upon interactions among immune cells. Specialized functions of CD4 and CD8 T cells were less dependent upon and often dampened by the presence of other PBMC populations. In particular, cytotoxic T cell effector function was variably affected with a component strictly dependent upon the direct stimulation of CD8 T cells in the absence of other PBMC. This observation may provide a roadmap for the interpretation of the discrepant biological activities of rIL-2 observed in distinct pathological conditions or treatment modalities.

There is an increasing interest in complementing conventional histopathologic evaluation with molecular tools that could increase the sensitivity and specificity of cancer staging for diagnostic and prognostic purposes. This study strove to identify cancer-specific markers for the molecular detection of a broad range of cancer types. We used 373 archival samples inclusive of normal tissues of various lineages and benign or malignant tumors (predominantly colon, melanoma, ovarian, and esophageal cancers). All samples were processed identically and cohybridized with an identical reference RNA source to a custom-made cDNA array platform. The database was split into training (n = 201) and comparable prediction (n = 172) sets. Leave-one-out cross-validation and gene pairing analysis identified putative cancer biomarkers overexpressed by malignant lesions independent of tissue of derivation. In particular, seven gene pairs were identified with high predictive power (87%) in segregating malignant from benign lesions. Receiver operator characteristic curves based on the same genes could segregate malignant from benign tissues with 94% accuracy. The relevance of this study rests on the identification of a restricted number of biomarkers ubiquitously expressed by cancers of distinct histology. This has not been done before. These biomarkers could be used broadly to increase the sensitivity and accuracy of cancer staging and early detection of locoregional or systemic recurrence. Their selective expression by cancerous compared with paired normal tissues suggests an association with the oncogenic process resulting in stable expression during disease progression when the presently used differentiation markers are unreliable. (Cancer Res 2006; 66(6): 2953-61)

A patient with metastatic cutaneous melanoma responsive to immunotherapy experienced several recurrences over a decade of observation. With each recurrence, biopsies were obtained and cell lines generated. A rare mutation of the beta-catenin gene and an unbalanced methylation of the androgen receptor were documented in all cell lines. Karyotyping and comparative genomic hybridization identified consistent genetic traits in spite of divergent phenotypes, suggesting that all the metastases were derived from the same primary tumor, although they were each probably not derived from the most recent previous metastasis in a sequential manner. Thus, metastatic melanoma recurs from a common progenitor cell and phenotypic changes occur around a central core of genetic stability. This observation may bear significance for the development of targeted anticancer therapies.