Measuring virus in biofluids is complicated by confounding biomolecules coisolated with viral nucleic acids. To address this, we developed an affinity-based microfluidic device for specific capture of intact severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Our approach used an engineered angiotensin-converting enzyme 2 to capture intact virus from plasma and other complex biofluids. Our device leverages a staggered herringbone pattern, nanoparticle surface coating, and processing conditions to achieve detection of as few as 3 viral copies per milliliter. We further validated our microfluidic assay on 103 plasma, 36 saliva, and 29 stool samples collected from unique patients with COVID-19, showing SARS-CoV-2 detection in 72% of plasma samples. Longitudinal monitoring in the plasma revealed our device’s capacity for ultrasensitive detection of active viral infections over time. Our technology can be adapted to target other viruses using relevant cell entry molecules for affinity capture. This versatility underscores the potential for widespread application in viral load monitoring and disease management.

Supplementary Table S1. Genes tested for Prostate CTC digital PCR Assay; Supplementary Table S2. Clinical information for prostate cancer patients and healthy donor subjects; Supplementary Table S3. Clinical information for patients enrolled on prospective trial of first-line abiraterone for mCRPC; Supplementary Table S4. Clinical information for patients with localized prostate cancer in radical prostatectomy study; Supplementary Table S5. Droplet digital PCR primers used in this study.

Methods describing microfluidic CTC enrichment, RNA extraction, amplification and detection, single cell and bulk RNA sequencing

Supplementary Figure S1. (A) Schematic of d-CTC assay. Whole blood is processed using the CTC-iChip, resulting in a bulk product highly enriched for intact CTCs (labeled green) but also contains WBCs (labeled red) and RBCs (unlabeled); Supplementary Figure S2. Graphs showing droplet digital PCR signal for each gene in 12 metastatic prostate cancer patients compared to 34 healthy male control subjects; Supplementary Figure S3. (A, B) Graphs of relationships between Prostate CTCM Score and serum PSA, and CTC droplet digital PCR KLK3 signal and serum PSA at the pre-treatment time point; Supplementary Figure S4. (A) Kaplan-Meier curves for radiographic progression-free survival (R-PFS) by CTC HOXB13 status at pretreatment; Supplementary Figure S5. (A) Heatmap of droplet digital PCR CTC signal after whole transcriptome amplification for blood samples from healthy donor controls arranged by sex and age, and from patients with clinically localized prostate cancer, arranged by D'Amico Risk Group; Supplementary Figure S6. (A) Box plots showing pre-operative leave-one-out cross validated (LOOCV) CTCL Score in clinically localized prostate cancer patients according to microscopic SVI or pelvic LN involvement identified at the time of radical prostatectomy

Microfluidic devices have been used for decades to isolate cells, viruses, and proteins using on-chip immunoaffinity capture using biotinylated antibodies, proteins, or aptamers. To accomplish this, the inner surface is modified to present binding moieties for the desired analyte. While this approach is successful in research settings, it is challenging to scale many surface modification strategies. Traditional polydimethylsiloxane (PDMS) devices can be effectively functionalized using silane-based methods; however, it requires high labor hours, equipment, and hazardous chemicals. Manufacture of microfluidic devices using plastics, including cyclic olefin copolymer (COC), allows chips to be mass produced, but most functionalization methods used with PDMS are not compatible with plastic. Herein, this work demonstrates how to deposit biotin onto the surface of a plastic microfluidic chips using aryl-diazonium. This method chemically bonds biotin to the surface, allowing for the addition of streptavidin nanoparticles to the surface. Nanoparticles increase the surface area of the chip and allow for proper capture moiety orientation. This process is faster, can be performed outside of a fume hood, is very cost-effective using readily available laboratory equipment, and demonstrates higher rates of capture. Additionally, this method allows for more rapid and scalable production of devices, including for diagnostic testing.

Cancer therapy often results in heterogeneous responses in different metastatic lesions in the same patient. Inter- and intratumor heterogeneity in signaling within various tumor compartments and its impact on therapy are not well characterized due to the limited sensitivity of single-cell proteomic approaches. To overcome this barrier, we applied single-cell mass cytometry with a customized 26-antibody panel to PTEN-deleted orthotopic prostate cancer xenograft models to measure the evolution of kinase activities in different tumor compartments during metastasis or drug treatment. Compared with primary tumors and circulating tumor cells (CTC), bone metastases, but not lung and liver metastases, exhibited elevated PI3K/mTOR signaling and overexpressed receptor tyrosine kinases (RTK) including c-MET protein. Suppression of c-MET impaired tumor growth in the bone. Intratumoral heterogeneity within tumor compartments also arose from highly proliferative EpCAM-high epithelial cells with increased PI3K and mTOR kinase activities coexisting with poorly proliferating EpCAM-low mesenchymal populations with reduced kinase activities; these findings were recapitulated in epithelial and mesenchymal CTC populations in patients with metastatic prostate and breast cancer. Increased kinase activity in EpCAM-high cells rendered them more sensitive to PI3K/mTOR inhibition, and drug-resistant EpCAM-low populations with reduced kinase activity emerged over time. Taken together, single-cell proteomics indicate that microenvironment- and cell state-dependent activation of kinase networks create heterogeneity and differential drug sensitivity among and within tumor populations across different sites, defining a new paradigm of drug responses to kinase inhibitors. SIGNIFICANCE:Single-cell mass cytometry analyses provide insights into the differences in kinase activities across tumor compartments and cell states, which contribute to heterogeneous responses to targeted therapies.

Deregulation of oncogenic signals in cancer triggers replication stress. Immediate early genes (IEGs) are rapidly and transiently expressed following stressful signals, contributing to an integrated response. Here, we find that the orphan nuclear receptor NR4A1 localizes across the gene body and 3' UTR of IEGs, where it inhibits transcriptional elongation by RNA Pol II, generating R-loops and accessible chromatin domains. Acute replication stress causes immediate dissociation of NR4A1 and a burst of transcriptionally poised IEG expression. Ectopic expression of NR4A1 enhances tumorigenesis by breast cancer cells, while its deletion leads to massive chromosomal instability and proliferative failure, driven by deregulated expression of its IEG target, FOS. Approximately half of breast and other primary cancers exhibit accessible chromatin domains at IEG gene bodies, consistent with this stress-regulatory pathway. Cancers that have retained this mechanism in adapting to oncogenic replication stress may be dependent on NR4A1 for their proliferation.

Extracellular vesicles (EVs) have emerged as promising candidates in biomarker discovery and diagnostics. Protected by the lipid bilayer, the molecular content of EVs in diverse biofluids are protected from RNases and proteases in the surrounding environment that may rapidly degrade targets of interests. Nonetheless, cryopreservation of EV-containing samples to -80°C may expose the lipid bilayer to physical and biological stressors which may result in cryoinjury and contribute to changes in EV yield, function, or molecular cargo. In the present work, we systematically evaluate the effect of cryopreservation at -80°C for a relatively short duration of storage (up to 12 days) on plasma- and media-derived EV particle count and/or RNA yield/quality, as compared to paired fresh controls. On average, we found that the plasma-derived EV concentration of stored samples decreased to 23% of fresh samples. Further, this significant decrease in EV particle count was matched with a corresponding significant decrease in RNA yield whereby plasma-derived stored samples contained only 47–52% of the total RNA from fresh samples, depending on the extraction method used. Similarly, media-derived EVs showed a statistically significant decrease in RNA yield whereby stored samples were 58% of the total RNA from fresh samples. In contrast, we did not obtain clear evidence of decreased RNA quality through analysis of RNA traces. These results suggest that samples stored for up to 12 days can indeed produce high-quality RNA; however, we note that when directly comparing fresh versus cryopreserved samples without cryoprotective agents there are significant losses in total RNA. Finally, we demonstrate that the addition of the commonly used cryoprotectant agent, DMSO, alongside greater control of the rate of cooling/warming, can rescue EVs from damaging ice formation and improve RNA yield.

ABSTRACTCancer therapy often results in heterogeneous responses in different metastatic lesions in the same patient. Inter- and intra-tumor heterogeneity in proteomic signaling within the various tumor compartments and its impact on therapy are not well characterized due to the limited sensitivity of single cell proteomic approaches. To overcome this barrier, we applied single cell mass cytometry with a customized 29-antibody panel [against cell states, receptor tyrosine kinases (RTK) and phosphoinositide 3-kinase/mammalian target of rapamycin (PI3K/mTOR)-, mitogen-activated protein kinase (MAPK)-, and cytokine-signaling] to PTEN-deleted orthotopic prostate cancer xenograft models to measure the evolution of kinase activities in different tumor compartments during metastasis and upon drug treatment. Compared with primary tumors and circulating tumor cells (CTCs), bone metastases but not lung and liver metastases exhibited elevated PI3K/mTOR signaling and RTKs including c-Met protein, which, when suppressed, impaired tumor growth in the bone. Intra-tumoral heterogeneity within tumor compartments also arises from highly proliferative EpCAMhighepithelial cells with increased PI3K and mTOR kinase activities co-existing with poorly proliferating EpCAMlowmesenchymal populations with reduced kinase activities, findings recapitulated in epithelial and mesenchymal CTC populations in metastatic prostate and breast cancer patients. Increased kinase activity in EpCAMhighcells rendered them more sensitive to PI3K/mTOR inhibition and drug resistant EpCAMlowpopulations with reduced kinase activity emerged over time. Taken together, single cell proteomics identified microenvironment- and cell state-dependent activation of kinase networks creating heterogeneity and differential drug sensitivity among and within tumor populations across different sites, defining a new paradigm of drug responses to kinase inhibitors.

Abstract A subset of patients with metastatic melanoma have sustained remissions following treatment with immune checkpoint inhibitors. However, analyses of pretreatment tumor biopsies for markers predictive of response, including PD-L1 expression and mutational burden, are insufficiently precise to guide treatment selection and clinical radiographic evidence of response on therapy may be delayed, leading to some patients receiving potentially ineffective but toxic therapy. Here, we developed a molecular signature of melanoma Circulating Tumor Cells (CTCs) to quantify early tumor response using blood-based monitoring. A quantitative 19-gene digital RNA signature (CTC-Score) applied to microfluidically-enriched CTCs robustly distinguishes melanoma cells, within a background of blood cells in reconstituted and in patient-derived (N=42) blood specimens. In a prospective cohort of 49 patients treated with immune checkpoint inhibitors, a decrease in CTC-Score within 7 weeks of therapy correlates with marked improvement in progression-free survival (Hazard Ratio (HR): 0.17, P=0.008) and overall survival (HR: 0.12, P=0.04). Thus, digital quantitation of melanoma CTC-derived transcripts enables serial noninvasive monitoring of tumor burden, supporting the rational application of immune checkpoint inhibition therapies. Citation Format: Xin Hong, Ryan J. Sullivan, Mark Kalinich, Tanya Kwan, Shiwei Pan, Joseph A. LiCausi, John D. Milner, Linda T. Nieman, Ben S. Wittner, Uyen Ho, Tianqi Chen, Ravi Kapur, Don Lawrence, Keith T. Flaherty, Lecia V. Sequist, Sridhar Ramaswamy, David T. Miyamoto, Michael Lawrence, Anita Giobbie-Hurder, Mehmet Toner, Kurt J. Isselbacher, Shyamala Maheswaran, Daniel A. Haber. Molecular signatures of circulating melanoma cells for monitoring early response to immune checkpoint therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-144.

Significance Identifying predictive biomarkers of therapeutic response for melanoma patients treated with immune checkpoint inhibitors is a major challenge. By combining microfluidic enrichment for melanoma circulating tumor cells (CTCs) together with RNA-based droplet digital PCR quantitation, we have established a highly sensitive and robust platform for noninvasive, blood-based monitoring of tumor burden. Serial monitoring of melanoma patients treated with immune checkpoint inhibitors shows rapid changes in CTC score, which precede standard clinical assessment and are highly predictive of long-term clinical outcome. Early on-treatment digital monitoring of CTC dynamics may thus help identify patients likely to benefit from immune checkpoint inhibition therapy.

Abstract Blood-based biomarkers are critical in metastatic prostate cancer, where characteristic bone metastases are not readily sampled, and they may enable risk stratification in localized disease. We established a sensitive and high-throughput strategy for analyzing prostate circulating tumor cells (CTC) using microfluidic cell enrichment followed by digital quantitation of prostate-derived transcripts. In a prospective study of 27 patients with metastatic castration-resistant prostate cancer treated with first-line abiraterone, pretreatment elevation of the digital CTCM score identifies a high-risk population with poor overall survival (HR = 6.0; P = 0.01) and short radiographic progression-free survival (HR = 3.2; P = 0.046). Expression of HOXB13 in CTCs identifies 6 of 6 patients with ≤12-month survival, with a subset also expressing the ARV7 splice variant. In a second cohort of 34 men with localized prostate cancer, an elevated preoperative CTCL score predicts microscopic dissemination to seminal vesicles and/or lymph nodes (P < 0.001). Thus, digital quantitation of CTC-specific transcripts enables noninvasive monitoring that may guide treatment selection in both metastatic and localized prostate cancer. Significance: There is an unmet need for biomarkers to guide prostate cancer therapies, for curative treatment of localized cancer and for application of molecularly targeted agents in metastatic disease. Digital quantitation of prostate CTC-derived transcripts in blood specimens is predictive of abiraterone response in metastatic cancer and of early dissemination in localized cancer. Cancer Discov; 8(3); 288–303. ©2018 AACR. See related commentary by Heitzer and Speicher, p. 269. This article is highlighted in the In This Issue feature, p. 253

Abstract The multiplicity of new therapies for breast cancer presents a challenge for treatment selection. We describe a 17-gene digital signature of breast circulating tumor cell (CTC)–derived transcripts enriched from blood, enabling high-sensitivity early monitoring of response. In a prospective cohort of localized breast cancer, an elevated CTC score after three cycles of neoadjuvant therapy is associated with residual disease at surgery (P = 0.047). In a second prospective cohort with metastatic breast cancer, baseline CTC score correlates with overall survival (P = 0.02), as does persistent CTC signal after 4 weeks of treatment (P = 0.01). In the subset with estrogen receptor (ER)–positive disease, failure to suppress ER signaling within CTCs after 3 weeks of endocrine therapy predicts early progression (P = 0.008). Drug-refractory ER signaling within CTCs overlaps partially with presence of ESR1 mutations, pointing to diverse mechanisms of acquired endocrine drug resistance. Thus, CTC-derived digital RNA signatures enable noninvasive pharmacodynamic measurements to inform therapy in breast cancer. Significance: Digital analysis of RNA from CTCs interrogates treatment responses of both localized and metastatic breast cancer. Quantifying CTC-derived ER signaling during treatment identifies patients failing to respond to ER suppression despite having functional ESR1. Thus, noninvasive scoring of CTC-RNA signatures may help guide therapeutic choices in localized and advanced breast cancer. Cancer Discov; 8(10); 1286–99. ©2018 AACR. This article is highlighted in the In This Issue feature, p. 1195

Circulating tumor cells (CTCs) are shed into the bloodstream by invasive cancers, but the difficulty inherent in identifying these rare cells by microscopy has precluded their routine use in monitoring or screening for cancer. We recently described a high-throughput microfluidic CTC-iChip, which efficiently depletes hematopoietic cells from blood specimens and enriches for CTCs with well-preserved RNA. Application of RNA-based digital PCR to detect CTC-derived signatures may thus enable highly accurate tissue lineage-based cancer detection in blood specimens. As proof of principle, we examined hepatocellular carcinoma (HCC), a cancer that is derived from liver cells bearing a unique gene expression profile. After identifying a digital signature of 10 liver-specific transcripts, we used a cross-validated logistic regression model to identify the presence of HCC-derived CTCs in nine of 16 (56%) untreated patients with HCC versus one of 31 (3%) patients with nonmalignant liver disease at risk for developing HCC (P < 0.0001). Positive CTC scores declined in treated patients: Nine of 32 (28%) patients receiving therapy and only one of 15 (7%) patients who had undergone curative-intent ablation, surgery, or liver transplantation were positive. RNA-based digital CTC scoring was not correlated with the standard HCC serum protein marker alpha fetoprotein (P = 0.57). Modeling the sequential use of these two orthogonal markers for liver cancer screening in patients with high-risk cirrhosis generates positive and negative predictive values of 80% and 86%, respectively. Thus, digital RNA quantitation constitutes a sensitive and specific CTC readout, enabling high-throughput clinical applications, such as noninvasive screening for HCC in populations where viral hepatitis and cirrhosis are prevalent.

Abstract Examination of circulating tumor cells (CTCs) holds the potential of offering a real-time non-invasive window into tumor biology. Information gleaned from CTC detection and characterization can be used for early cancer detection, choice of therapy decisions and long-term monitoring for disease recurrence and the emergence of drug resistance mechanisms. However, technical difficulties with CTC isolation and the inherent limitations of imaging-based analysis have hindered the broad clinical use of CTCs as biomarkers. To overcome these concerns, we have combined our unbiased microfluidic CTC enrichment technology, the iChip, with a highly sensitive and specific multi-gene RNA-based biomarker panel to develop an assay that detects CTC signatures in patient blood samples in a high throughput and quantitative fashion. This assay was applied to a cohort of breast cancer patients, including women with both localized and metastatic disease. It successfully identified CTC signal in 50-70% of metastatic and 20-40% of localized pretreatment patient samples. Patient CTC scores, assigned based on a multi-marker prediction algorithm, correlated with cancer stage and grade, but not with hormone receptor status, suggesting the applicability of the assay to a wide range of breast cancer subtypes. To determine if CTC scoring was useful for disease monitoring, we performed monthly blood collection from metastatic patients starting a new line of treatment. CTC score as early as one month after initiation of therapy, but not at pretreatment, was predictive of progression-free survival and treatment outcome, suggesting that real-time kinetic changes in CTC signatures are more clinically informative that one-time static evaluation of their presence in a given sample. In addition, a subset of high-risk genes showed expression patterns over the course of treatment that highly correlated with disease recurrence. This observation indicates that a multi-marker panel can parse out dynamic changes in cancer gene expression programs and provide a molecular insight into treatment responses that are not achievable by assays built around one or few markers. In conclusion, our novel RNA-based method of identifying CTC signatures in liquid biopsies provides a sensitive platform for breast cancer detection and monitoring that goes beyond CTC enumeration. It offers a non-invasive quantitative molecular characterization of tumor gene expression that can be used to guide informed clinical decisions in both standard course of care and clinical trial settings. Citation Format: Tanya T. Kwan, Aditya Bardia, Tilak Sundaresan, Laura Spring, Mark Kalinich, David Miyamoto, Xin Hong, Joseph LiCausi, Uyen Ho, Sarah Javaid, Erin Silva, Lecia Sequist, Shyamala Maheswaran, Daniel Haber. A novel RNA-based assay for the detection and monitoring of circulating tumor cell signatures in 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 1736. doi:10.1158/1538-7445.AM2017-1736