Abstract: Molecular drivers underlying bone metastases in human cancer are not well understood, in part due to constraints in bone tissue sampling. Here, RNA sequencing was performed of circulating tumor cells (CTC) isolated from blood samples of women with metastatic estrogen receptor (ER)+ breast cancer, comparing cases with progression in bone versus visceral organs. Among the activated cellular pathways in CTCs from bone-predominant breast cancer is androgen receptor (AR) signaling. AR gene expression is evident, as is its constitutively active splice variant AR-v7. AR expression within CTCs is correlated with the duration of treatment with aromatase inhibitors, suggesting that it contributes to acquired resistance to endocrine therapy. In an established breast cancer xenograft model, a bone-tropic derivative displays increased AR expression, whose genetic or pharmacologic suppression reduces metastases to bone but not to lungs. Together, these observations identify AR signaling in CTCs from women with bone-predominant ER+ breast cancer, and provide a rationale for testing androgen inhibitors in this subset of patients. Implications: This study highlights a role for the AR in breast cancer bone metastasis, and suggests that therapeutic targeting of the AR may benefit patients with metastatic breast cancer. Mol Cancer Res; 16(4); 720–7. ©2018 AACR.

Abstract: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal adult malignancy due to the propensity of this disease to metastasize. Circulating tumor cells (CTCs) are thought to be enriched for cells with metastatic potential and their characterization offers a means to understand the biological underpinnings of the distal spread of cancer. However, CTCs are rare cells in the blood and their isolation has posed a signifcant technological challenge. Multiple platforms have emerged in capturing these cells with most relying on a positive antibody based selection process (e.g. EpCAM). Our initial molecular characterization of pancreatic CTCs in the genetically engineered LSL-KrasG12D, Trp53flox/flox or +, Pdx1-Cre (KPC) mouse model utilized a microfluidic anti-EpCAM device followed by RNA sequencing to identify aberrant non-canonical WNT signaling in CTC populations (Yu M*, Ting DT*, et al. Nature 2012). Wnt2 was found to increase metastatic potential by enhancing anoikis resistance, a requirement of CTC survival, through activation of Tak1 kinase. However, this study was limited in only providing a partial CTC signature given the variable purity of these purified cell populations and analysis of bulk CTC populations could not provide the resolution to truly assess heterogeneity of these cells. Furthermore, a recent study has shown that cells from the mouse model may disseminate into circulation at an early point in PDAC development through epithelial-to-mesenchymal transition (EMT), which would generate CTCs with very low EpCAM expression and therefore would be missed in an EpCAM based capture device (Rhim AD et al. Cell 2012). We have overcome these barriers by employing a novel microfluidic isolation device to capture high numbers of CTCs that can be isolated as single cells (Ozkumur E*, Shah AM*, et al. Science Translational Medicine 2013). This device achieves high efficiency negative depletion of normal blood cells providing an enriched population of CTCs in solution that are not biased by a particular extracellular epitope and without any antibody interactions that could affect expression profiles. This has provided a method to truly understand the key transcriptional programs that differentiate CTCs from their primary tumors on an isogenic mouse background. Five tumor-bearing KPC mice generated a total of 168 single CTCs that were morphologically intact and subjected to a modified single cell RNA sequencing protocol (Tang F et al., Nature Protocols 2010). A total of 75 (45%) of these single CTCs were of sufficient quality for RNA sequencing indicating that the majority (55%) of intact CTCs selected likely had lost viability in the process of hematogenous transit. Single cell RNA-sequencing was also performed on 12 normal leukocytes (WBCs) from a control mouse, 12 mouse embryonic fibroblasts (MEFs), 16 single cells from the mouse NB508 pancreatic cancer cell line, and 34 (min 8 replicates) samples from primary tumors matched to the CTCs. Unsupervised hierarchical clustering of single cell samples demonstrated clear separation of MEFs, the NB508 pancreatic cancer cell line, and normal WBCs supporting the technical validity of the sequencing approach. Analysis of candidate CTCs identified three major CTC clusters, which were all distinct form matched primary tumors as well as from the NB508 cancer cell line. The must abundant CTC cluster comprised 41 of 75 cells (55%) and was defined by presence of epithelial markers (Krt7, Krt8, Krt18, Krt19) consistent with a “classical” CTC phenotype (CTC-c). The second CTC cluster was defined by enrichment of platelet markers CD41 (Itga2b) and CD61 (Itgb3) (CTC-plt) and a third having enrichment of cellular proliferation genes including Mki67 (CTC-pro). Single cell heterogeneity was assessed by intra-cluster correlation coeffficients, where lower values reflect higher heterogeneity. Not surprisingly, single cell heterogeneity was much higher in CTCs (mean correlation coefficient 0.42, 95% CI 0.36-0.47) compared to cancer cell lines (mean 0.86, 95% CI 0.80-0.91, p-value 1.2 x 10-15), but was notably similar comparing CTCs to single primary tumor cells (mean 0.38, 95% CI 0.28-0.47). Focusing on the dominant CTC-c cells, we used a non-parametric differential gene expression analysis including a rank product (RP) methodology suitable for large variations in absolute transcript levels found in single cell expression data (Breitling R et al. FEBS Letters 2004). Using a stringent FDR of ≤ 0.01, CTC-c cells had 878 genes with increased expression and 774 genes with reduced expression when compared with matched primary tumors. CTC-c cells were enriched for MAPK, as well as WNT, TGF-β, Neurotrophin, Toll-like receptor, and B-cell receptor signaling pathways. Analysis of a panel of EMT genes with significant differential expression revealed that CTC-c cells were in a biphenotypic state with universal loss of the epithelial markers E-cadherin (Cdh1) and Muc1, while the mesenchymal genes Cdh11 and Vim were found to be expressed much more heterogeneously amongst individual CTCs. Proposed pancreatic cancer stem cell genes were also evaluated and the Aldh1a1 and Aldh1a2 genes were found to be significantly enriched in CTC-c. Expression of both Aldh1a1 and Aldh1a2 in matched primary tumors was done through RNA in situ hybridization (RNA-ISH) revealing a heterogeneous distribution of these stem cell genes in both the stromal and epithelial compartments of the tumor. This highlighted the potential relevance of these stem cell markers in tumor cells dynamically shifting between epithelial and non-epithelial states. To provide further insight into the potential region from which CTCs emanate from the primary tumor, we selected the most highly enriched CTC transcripts found in ≥ 90% of all classical CTCs. Three genes met these criteria, which were decorin (Dcn), insulin-like growth factor binding protein 5 (Igfbp5), and Kruppel-like factor 4 (Klf4). Each of these genes has been previously implicated in pancreatic cancer development and were evaluated by RNA-ISH in primary tumor specimens to determine if they colocalized in particular tumor cells. Dcn is an extracellular matrix proteoglycan known to be expressed in a wide range of tumor stroma and by RNA-ISH was found primarily in the stromal elements of the tumor. However, both Igfbp5 and Klf4 were found to be focally expressed in cells at the epithelial-stromal interface. Although these genes are co-expressed in a minority of primary tumor cells, they are co-expressed at high levels in 85% of all classical CTCs. Together with the mixed epithelial/mesenchymal marks and enrichment of Aldh1a2 cells in stromal elements, these data point to the majority of viable CTCs emanating from the epithelial/stromal interface. In summary, we have successfully purified individual pancreatic CTCs using a novel microfluidic device and provided the first comprehensive single cell transcriptome analysis of these rare but exceptional cells. Three major classes of CTCs have been identified that would not have been possible without a single cell approach and we have characterized the major pathways that define these different subsets. The most abundant CTCs were found to have robust expression of keratin genes and are defined by a mixed E/M state with enrichment of Aldh1a1 and Aldh1a2 stem cell genes. These classical CTCs are marked by the co-expression of Igfbp5 and Klf4, which appear to localize to the epithelial-stromal interface in primary tumors. Ultimately, CTC cultures and functional testing will determine the contribution of these genes to CTC metastatic potential. This deep analysis of CTCs at single cell resolution has provided new biological insight into the metastatic cascade that will inform the development of novel therapies to treat this deadly disease. Citation Format: David T. Ting, Ben S. Wittner, Ajay M. Shah, David T. Miyamoto, Brian W. Brannigan, Kristina Xega, Jordan Ciciliano, Olivia C. MacKenzie, Julie Trautwein, Mohammad Shahid, Haley L. Ellis, Na Qu, Nabeel Bardeesy, Miguel N. Rivera, Ravi Kapur, Sridhar Ramaswamy, Toshi Shioda, Mehmet Toner, Shyamala Maheswaran, Daniel A. Haber. Diversity of circulating tumor cells in a mouse pancreatic cancer model identified by single cell RNA sequencing. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr NG04. doi:10.1158/1538-7445.AM2014-NG04

Abstract: The metastatic spread of breast cancer, typically to bone, lung, liver and brain, accounts for the vast majority of cancer-related deaths. Breast cancer metastases are thought to be derived primarily from individual migratory cancer cells, reaching distant sites through the bloodstream and initiating proliferation within distant organs. In addition to these single circulating tumor cells (CTCs), CTC-clusters have been detected in the blood of patients with cancer. In patients with breast cancer, we find that presence of such CTC-clusters is correlated with decreased progression-free survival. To study their functional role, we used mouse models, demonstrating that breast cancer cells injected intravascularly as clusters are more prone to survive and colonize the lungs than similarly injected single cancer cells. Primary orthotopic mammary tumors comprised of differentially tagged cells give rise to oligoclonal CTC-clusters, with 40-fold increased metastatic potential to the lung, compared with single CTCs. Using in vivo flow cytometry, we show that CTC-clusters are rapidly cleared from peripheral vessels, consistent with their trapping in small capillaries. Together, our observations suggest that primary tumor cells break off into the vasculature as CTC-clusters, and exhibit greatly enhanced metastatic propensity. Citation Format: Nicola Aceto, Aditya Bardia, Joel A. Spencer, Ben S. Wittner, Min Yu, Maria C. Donaldson, Adam Pely, Amanda Engstrom, Huili Zhu, Brian W. Brannigan, Ravi Kapur, Shannon L. Stott, Toshi Shioda, Sridhar Ramaswamy, David T. Ting, Charles P. Lin, Mehmet Toner, Daniel A. Haber, Shyamala Maheswaran. Circulating tumor cell clusters are precursors of breast cancer metastasis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-192. doi:10.1158/1538-7445.AM2014-LB-192

Abstract: Prostate cancer is initially responsive to androgen deprivation, but the effectiveness of androgen receptor (AR) inhibitors in recurrent disease is variable. Biopsy of bone metastases is challenging; hence, sampling circulating tumor cells (CTCs) may reveal drug-resistance mechanisms. We established single-cell RNA-sequencing (RNA-Seq) profiles of 77 intact CTCs isolated from 13 patients (mean six CTCs per patient), by using microfluidic enrichment. Single CTCs from each individual display considerable heterogeneity, including expression of AR gene mutations and splicing variants. Retrospective analysis of CTCs from patients progressing under treatment with an AR inhibitor, compared with untreated cases, indicates activation of noncanonical Wnt signaling ( P = 0.0064). Ectopic expression of Wnt5a in prostate cancer cells attenuates the antiproliferative effect of AR inhibition, whereas its suppression in drug-resistant cells restores partial sensitivity, a correlation also evident in an established mouse model. Thus, single-cell analysis of prostate CTCs reveals heterogeneity in signaling pathways that could contribute to treatment failure.

Abstract: Isolation of circulating tumor cells (CTCs) in the blood allows noninvasive tumor sampling from patients with cancer. Molecular analysis of single CTCs may uncover heterogeneous cellular pathways that underlie disease progression and resistance to therapy. Using a microfluidic device, we isolated individual CTCs from patients with metastatic prostate cancer. Single candidate CTCs were micromanipulated after cell surface staining for epithelial (EpCAM) and mesenchymal (CDH11) markers, and then subjected to single cell RNA-sequencing. Digital gene expression profiles of lineage-confirmed CTCs were compared with each other, with primary prostate tumors, and with annotated markers of cellular signaling pathways. Single prostate CTCs displayed considerable heterogeneity in transcriptional profiles, but clustered according to patient of origin, indicating higher diversity in CTCs across different individuals (mean correlation 0.10 for CTCs within patients vs. 0.0014 for CTCs across patients, P=2.0x10E-11). Compared to primary tumors, CTCs were significantly enriched in 37 molecular pathways (FDR & lt;0.1), with the majority implicated in growth factor, cell adhesion, and hormone signaling. Gene mutations and alternative splice variants of the Androgen Receptor (AR) gene were rare in primary prostate tumors and CTCs from untreated patients, but were prevalent in patients with castration-resistant prostate cancer. Distinct AR variants, including AR-V7, were present within different cells of individual patients, as well as within individual CTCs. Together, single cell molecular analysis of human CTCs points to multiple mechanisms of drug resistance in advanced prostate cancer, and suggests the role of heterogeneous signaling pathways that cooperate with co-existing abnormalities in AR in mediating disease progression. Citation Format: David T. Miyamoto, Yu Zheng, Ben S. Wittner, Richard J. Lee, Huili Zhu, Katherine T. Broderick, Rushil Desai, Brian W. Brannigan, Kshitij S. Arora, Douglas M. Dahl, Lecia V. Sequist, Matthew R. Smith, Ravi Kapur, Chin-Lee Wu, Toshi Shioda, Sridhar Ramaswamy, David T. Ting, Mehmet Toner, Shyamala Maheswaran, Daniel A. Haber. Single cell RNA-sequencing of circulating tumor cells. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Integrating Clinical Genomics and Cancer Therapy; Jun 13-16, 2015; Salt Lake City, UT. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(1_Suppl):Abstract nr IA09.