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DNA-FISH analysis of non-EGFR-amplified GBM CTCs.

Immunohistologic and DNA-FISH analysis of tumor cells and CTCs from a patient with metastatic GBM.

Methods and Supplementary Figure Legends

STEAM cocktail staining specifically identifies GBM cells.

Clinical association of CTC frequency

Unsupervised hierarchical clustering analysis of patient and xenograft single cell qRT-PCR expression data.

GBM8 and GBM24 growth in culture.

Tumor genotypes and CTC frequency

Purpose: Lung cancer stem cells (CSC) with elevated aldehyde dehydrogenase (ALDH) activity are self-renewing, clonogenic, and tumorigenic. The purpose of our study is to elucidate the mechanisms by which lung CSCs are regulated.Experimental Design: A genome-wide gene expression analysis was performed to identify genes differentially expressed in the ALDH+ versus ALDH− cells. RT-PCR, Western blot analysis, and Aldefluor assay were used to validate identified genes. To explore the function in CSCs, we manipulated their expression followed by colony and tumor formation assays.Results: We identified a subset of genes that were differentially expressed in common in ALDH+ cells, among which ALDH1A3 was the most upregulated gene in ALDH+ versus ALDH− cells. shRNA-mediated knockdown of ALDH1A3 in non–small cell lung cancer (NSCLC) resulted in a dramatic reduction in ALDH activity, clonogenicity, and tumorigenicity, indicating that ALDH1A3 is required for tumorigenic properties. In contrast, overexpression of ALDH1A3 by itself it was not sufficient to increase tumorigenicity. The ALDH+ cells also expressed more activated STAT3 than ALDH− cells. Inhibition of STAT3 or its activator EZH2 genetically or pharmacologically diminished the level of ALDH+ cells and clonogenicity. Unexpectedly, ALDH1A3 was highly expressed in female, never smokers, well-differentiated tumors, or adenocarcinoma. ALDH1A3 low expression was associated with poor overall survival.Conclusions: Our data show that ALDH1A3 is the predominant ALDH isozyme responsible for ALDH activity and tumorigenicity in most NSCLCs, and that inhibiting either ALDH1A3 or the STAT3 pathway are potential therapeutic strategies to eliminate the ALDH+ subpopulation in NSCLCs. Clin Cancer Res; 20(15); 4154–66. ©2014 AACR.

PDF file - 465K, Supplementary Table S1. The correlation between ALDH1A3 protein expression and percentage of ALDH+ cells in NSCLC lines (Data for Figure 2D). Supplementary Table S2. Incidence of xenograft tumors derived from control or ALDH1A3 knockdown lung cancer cells. Supplementary Table S3. The association between ALDH1A3 protein expression and clinicopathological variables for 455 NSCLC patient samples. Figure S1. ALDH profiling of 8 NSCLC lines using the flow cytometry based Aldefluor assay. Figure S2. Anchorage dependent colony formation efficiency was determined by growing serial dilutions of sorted ALDH+ and ALDH- cells for 2 weeks in liquid culture. Figure S3. A, ALDH1A3 protein expression in a set of NSCLC lines was detected by western blots. The corresponding percentage of ALDH+ cells was analyzed by Aldefluor assay. B, western blot showed ALDH1A1 protein expression in certain HBEC and NSCLC lines. C, ALDH1A3 was knocked down using three siRNAs in a panel of NSCLC lines with different driver mutations followed by liquid colony formation assays. RT-PCR was performed to examine ALDH1A3 mRNA expression. Figure S4. Ectopic expression of ALDH1A3 does not affect tumorigenicity of lung cancer cells.

Supplementary tables S1-S3. Supplementary Table S1: Institutional Tumor Genotyping Strategies. Supplementary Table S2: T790M Genotype by Modality. Supplementary Table S3: EGFR Primary Mutation Detection.

Purpose: The T790M gatekeeper mutation in the EGFR is acquired by some EGFR-mutant non–small cell lung cancers (NSCLC) as they become resistant to selective tyrosine kinase inhibitors (TKI). As third-generation EGFR TKIs that overcome T790M-associated resistance become available, noninvasive approaches to T790M detection will become critical to guide management.Experimental Design: As part of a multi-institutional Stand-Up-To-Cancer collaboration, we performed an exploratory analysis of 40 patients with EGFR-mutant tumors progressing on EGFR TKI therapy. We compared the T790M genotype from tumor biopsies with analysis of simultaneously collected circulating tumor cells (CTC) and circulating tumor DNA (ctDNA).Results: T790M genotypes were successfully obtained in 30 (75%) tumor biopsies, 28 (70%) CTC samples, and 32 (80%) ctDNA samples. The resistance-associated mutation was detected in 47% to 50% of patients using each of the genotyping assays, with concordance among them ranging from 57% to 74%. Although CTC- and ctDNA-based genotyping were each unsuccessful in 20% to 30% of cases, the two assays together enabled genotyping in all patients with an available blood sample, and they identified the T790M mutation in 14 (35%) patients in whom the concurrent biopsy was negative or indeterminate.Conclusions: Discordant genotypes between tumor biopsy and blood-based analyses may result from technological differences, as well as sampling different tumor cell populations. The use of complementary approaches may provide the most complete assessment of each patient's cancer, which should be validated in predicting response to T790M-targeted inhibitors. Clin Cancer Res; 22(5); 1103–10. ©2015 AACR.

Supplementary Figure 7 from Aldehyde Dehydrogenase Activity Selects for Lung Adenocarcinoma Stem Cells Dependent on Notch Signaling

Macrophages are an abundant inflammatory cell type in the tumor microenvironment that can contribute to tumor growth and metastasis. Macrophage recruitment into tumors is mediated by multiple cytokines, including vascular endothelial growth factor (VEGF), which is thought to function primarily through VEGF receptor (VEGFR) 1 expressed on macrophages. Macrophage infiltration is affected by VEGF inhibition. We show that selective inhibition of VEGFR2 reduced macrophage infiltration into orthotopic pancreatic tumors. Our studies show that tumor-associated macrophages express VEGFR2. Furthermore, peritoneal macrophages from tumor-bearing animals express VEGFR2, whereas peritoneal macrophages from non–tumor-bearing animals do not. To our knowledge, this is the first time that tumor-associated macrophages have been shown to express VEGFR2. Additionally, we found that the cytokine pleiotrophin is sufficient to induce VEGFR2 expression on macrophages. Pleiotrophin has previously been shown to induce expression of endothelial cell markers on macrophages and was present in the microenvironment of orthotopic pancreatic tumors. Finally, we show that VEGFR2, when expressed by macrophages, is essential for VEGF-stimulated migration of tumor-associated macrophages. In summary, tumor-associated macrophages express VEGFR2, and selective inhibition of VEGFR2 reduces recruitment of macrophages into orthotopic pancreatic tumors. Our results show an underappreciated mechanism of action that may directly contribute to the antitumor activity of angiogenesis inhibitors that block the VEGFR2 pathway. [Cancer Res 2008;68(11):4340–6]

Supplementary Figure 6 from Aldehyde Dehydrogenase Activity Selects for Lung Adenocarcinoma Stem Cells Dependent on Notch Signaling

Supplementary Figure 1 from Aldehyde Dehydrogenase Activity Selects for Lung Adenocarcinoma Stem Cells Dependent on Notch Signaling

Supplementary Figure 3 from Aldehyde Dehydrogenase Activity Selects for Lung Adenocarcinoma Stem Cells Dependent on Notch Signaling

Supplementary Table 4 from Aldehyde Dehydrogenase Activity Selects for Lung Adenocarcinoma Stem Cells Dependent on Notch Signaling

Supplementary Figure 1 from Vascular Endothelial Growth Factor Receptor 2 Mediates Macrophage Infiltration into Orthotopic Pancreatic Tumors in Mice

Supplementary Table 3 from Aldehyde Dehydrogenase Activity Selects for Lung Adenocarcinoma Stem Cells Dependent on Notch Signaling

Supplementary Figure and Table Legends from Aldehyde Dehydrogenase Activity Selects for Lung Adenocarcinoma Stem Cells Dependent on Notch Signaling

Steroid receptor coactivator-3 (SRC-3) is a histone acetyltransferase and nuclear hormone receptor coactivator, located on 20q12, which is amplified in several epithelial cancers and well studied in breast cancer. However, its possible role in lung cancer pathogenesis is unknown. We found SRC-3 to be overexpressed in 27% of non–small cell lung cancer (NSCLC) patients (n = 311) by immunohistochemistry, which correlated with poor disease-free (P = 0.0015) and overall (P = 0.0008) survival. Twenty-seven percent of NSCLCs exhibited SRC-3 gene amplification, and we found that lung cancer cell lines expressed higher levels of SRC-3 than did immortalized human bronchial epithelial cells (HBEC), which in turn expressed higher levels of SRC-3 than did cultured primary human HBECs. Small interfering RNA–mediated downregulation of SRC-3 in high-expressing, but not in low-expressing, lung cancer cells significantly inhibited tumor cell growth and induced apoptosis. Finally, we found that SRC-3 expression is inversely correlated with gefitinib sensitivity and that SRC-3 knockdown results in epidermal growth factor receptor tyrosine kinase inhibitor–resistant lung cancers becoming more sensitive to gefitinib. Taken together, these data suggest that SRC-3 may be an important oncogene and therapeutic target for lung cancer. Cancer Res; 70(16); 6477–85. ©2010 AACR.

Supplementary Figures 1-3, Tables 1-3 from Steroid Receptor Coactivator-3 Expression in Lung Cancer and Its Role in the Regulation of Cancer Cell Survival and Proliferation

Supplementary Figure 5 from Aldehyde Dehydrogenase Activity Selects for Lung Adenocarcinoma Stem Cells Dependent on Notch Signaling

Supplementary Figure 4 from Aldehyde Dehydrogenase Activity Selects for Lung Adenocarcinoma Stem Cells Dependent on Notch Signaling

Aldehyde dehydrogenase (ALDH) is a candidate marker for lung cancer cells with stem cell-like properties. Immunohistochemical staining of a large panel of primary non–small cell lung cancer (NSCLC) samples for ALDH1A1, ALDH3A1, and CD133 revealed a significant correlation between ALDH1A1 (but not ALDH3A1 or CD133) expression and poor prognosis in patients including those with stage I and N0 disease. Flow cytometric analysis of a panel of lung cancer cell lines and patient tumors revealed that most NSCLCs contain a subpopulation of cells with elevated ALDH activity, and that this activity is associated with ALDH1A1 expression. Isolated ALDH+ lung cancer cells were observed to be highly tumorigenic and clonogenic as well as capable of self-renewal compared with their ALDH− counterparts. Expression analysis of sorted cells revealed elevated Notch pathway transcript expression in ALDH+ cells. Suppression of the Notch pathway by treatment with either a γ-secretase inhibitor or stable expression of shRNA against NOTCH3 resulted in a significant decrease in ALDH+ lung cancer cells, commensurate with a reduction in tumor cell proliferation and clonogenicity. Taken together, these findings indicate that ALDH selects for a subpopulation of self-renewing NSCLC stem-like cells with increased tumorigenic potential, that NSCLCs harboring tumor cells with ALDH1A1 expression have inferior prognosis, and that ALDH1A1 and CD133 identify different tumor subpopulations. Therapeutic targeting of the Notch pathway reduces this ALDH+ component, implicating Notch signaling in lung cancer stem cell maintenance. Cancer Res; 70(23); 9937–48. ©2010 AACR.

Supplementary Figure 2 from Aldehyde Dehydrogenase Activity Selects for Lung Adenocarcinoma Stem Cells Dependent on Notch Signaling

Supplementary Table 2 from Aldehyde Dehydrogenase Activity Selects for Lung Adenocarcinoma Stem Cells Dependent on Notch Signaling

Supplementary Table 1 from Aldehyde Dehydrogenase Activity Selects for Lung Adenocarcinoma Stem Cells Dependent on Notch Signaling

Supplementary Figure 2 from Vascular Endothelial Growth Factor Receptor 2 Mediates Macrophage Infiltration into Orthotopic Pancreatic Tumors in Mice

Individual comorbidities have distinct contributions to nonrelapse mortality (NRM) following allogeneic hematopoietic cell transplantation (allo-HCT). We studied the impact of comorbidities individually and in combination in a single-center cohort of 573 adult patients who underwent CD34-selected allo-HCT following myeloablative conditioning. Pulmonary disease, moderate to severe hepatic comorbidity, cardiac disease of any type, and renal dysfunction were associated with increased NRM in multivariable Cox regression models. A Simplified Comorbidity Index (SCI) composed of the 4 comorbidities predictive of NRM, as well as age >60 years, stratified patients into 5 groups with a stepwise increase in NRM. NRM rates ranged from 11.4% to 49.9% by stratum, with adjusted hazard ratios of 1.84, 2.59, 3.57, and 5.38. The SCI was also applicable in an external cohort of 230 patients who underwent allo-HCT with unmanipulated grafts following intermediate-intensity conditioning. The area under the receiver operating characteristic curve (AUC) of the SCI for 1-year NRM was 70.3 and 72.0 over the development and external-validation cohorts, respectively; corresponding AUCs of the Hematopoietic Cell Transplantation–specific Comorbidity Index (HCT-CI) were 61.7 and 65.7. In summary, a small set of comorbidities, aggregated into the SCI, is highly predictive of NRM. The new index stratifies patients into distinct risk groups, was validated in an external cohort, and provides higher discrimination than does the HCT-CI.

“The Communist Party of China shall…expand the broadest possible patriotic united front…to strengthen the unity of all the Chinese people, including…overseas Chinese.” This passage from the 1956 Constitution of the Chinese Communist Party illustrates China’s expansionary international active measures which are, and have always been, the bedrock of Chinese foreign policy from its earliest days through today. Academics such as Peter Perdue have shown that China pursued continental consolidation and westward expansion in a manner far more consistent with the United States’ eventual hegemony over North America than any stylized version of a peaceful rise. This dynamic was true not only during early periods such as the aptly named Warring States period and subsequent dynasties, but also during periods where other historians such as Chung-in Moon and Seung-won Suh have argued for a “Confucian Long Peace” driven by the unique characteristics of China, in particular during the Qing Dynasty (1644-1912) prior to western intervention during the Opium Wars (1839-1860). Active measures were a core part of this story over a period of thousands of years. Inscriptions show intelligence operations during the middle Shang period (1200 BCE), and significant use of agents during the Spring and Autumn period (722-481 BCE), culminating in their aggressive deployment during the Warring States period (403-221 BCE). Their centrality was subsequently memorialized in Sunzi’s Art of War. These tactics included measures that will sound familiar to readers in the present day, as Jie Xuan wrote during the Ming Dynasty: “Creating forged letters is clandestine written action. Holding discussions that stupefy the enemy is clandestine civil action. Creating prophetic verses that circulate Ultimate among the people is clandestine action by prophecy”. Our opening quote helps to illustrate that the centrality of these policies continued under the Chinese Communist Party (CCP), since its very inception in 1921. This expansionary approach, and these active measures, continued unabated. This reality is not fully recognized by foreign policy professionals analyzing active measures today, who remain focused almost exclusively on Russian, Iranian, and North Korean efforts. It is the equivalent of the boardwalk magician, leveraging the sound and fury created by the right hand, while the left hand silently and slowly picks your pocket. Media and policy attention has been solely devoted to the sound and fury, while the slow and constant efforts of China have been largely ignored. Put another way, consensus has focused on the louder visible social networks such as Facebook leveraged by Russia and Iran, while ignoring the pervasive silent social network that is China’s United Front and its active measures. Global perception must catch up to the historical reality of China. This paper will explore perception gaps that exist regarding 1) China’s historically based expansionist nature (focusing on the period of the Qing dynasty prior to the Opium Wars, and the “peaceful rise” narrative championed for political expediency by the CCP), as well as 2) the significant evolution of United Front active measures from Mao Tse Tung, Deng Xiaoping, Hu Jintao, and on to the present day under Xi Jinping. Mainstream consensus thinking regarding China, at least until 20-25 years ago, positioned it as a nation and government primarily focused on domestic control, with a long history of non-expansionary behavior. This view has been in place since the early days of Western analysis of the Middle Kingdom, back to the 1930s when John Fairbank walked the streets of Peiping, and provided the scholarly foundation on which much of western thought regarding China has been built. Academics began to reconsider this conceptualization by the mid-1990s. Properly framing this reality is one of the most important questions facing International Relations scholars and foreign policy professionals today, as it cuts to the primary drivers and motivation of Chinese foreign policy efforts, and the tools utilized to advance them. This paper will show that China is, in my nomenclature, “Just Another Country” (JAC), and that the “Confucian Long Peace” was a politically motivated historical fallacy. It will then illustrate the importance of this misunderstanding, leveraging an analysis of the evolution of Chinese active measures campaigns under the United Front banner from their inception as the First United Front between the CCP and the Kuomintang (KMT) against warlords and imperialists (1923-1927), to the Second United Front to expel Japan (1937-1941), to a United Front against the Kuomintang in 1946, to a pivot by Mao to launch international United Front Activities beginning in South East Asia in 1948, to their strengthening under Xi. This history shows that an expansionist China leveraged active measures, albeit “with Chinese characteristics”, at least as aggressively as countries receiving far more publicity and government discussion time, such as Russia, Iran, or North Korea. This reality has far reaching implications for understanding Chinese foreign policy, which I argue deserves more policy and academic consideration relative to that devoted to the sound and fury of Russian measures. This paper is organized into three sections. The first will provide an overview of the historical Fairbank model of understanding China and the more recent challenge to this thinking led by scholars such as Perdue. The second will illustrate the “Chinese characteristics” of active measures through a brief history of United Front activities in China as adopted from the Soviet Union and their subsequent evolution in China under Mao and Deng and most particularly under the current Xi Government. The third and final section will provide an overall summary as well as a discussion regarding the differential goals at play for episodic disinformation campaigns vs. ongoing operating environment focused United Front strategies.

This paper compares the use of populism to control the middle class during periods of economic turbulence in the Soviet Union, its satellite states during the end of the Cold War, and Deng’s China. These case studies show anecdotal evidence that falling economic expectations coincide with rising populism.We next survey academic literature describing economic and social drivers of populism as well as the relationship between populism and nationalism. This survey is leveraged to construct a critique of existing attempts to quantify the drivers of populism, examining both supply and demand models. We attempt to add to this discussion by constructing a model tracking macroeconomic variables across time series data available from the Global Populism Database. We find meaningful inverse relationships between measures of supply side populism as measured in national leader speeches and Real and Nominal GDP, and direct relationships to GINI coefficients and Unemployment.Post COVID economic forecasts show a step down in global growth. This step down is forecast to be most extreme in India, the Philippines and Thailand, indicating the highest chance for rising populism. China, by contrast, is forecast to see a strong recovery, which when combined with stable middle class share of income trends suggest that populism / nationalism fears in China may be overblown. We leverage our prior case studies to understand the actors that might seek to use nationalism in these countries, and for what purposes.Incremental research questions include the development of fuller populism data sets allowing for deeper statistical analysis, work to understand if economic drivers impact right wing vs. left wing populism differently, as well as work to ascertain a feedback loop between falling economic conditions driving populism, and the economic efficacy of populist policies (and if these vary with left vs. right wing populist policies).