Early detection of pancreatic ductal adenocarcinoma (PDAC) remains elusive. Precursor lesions of PDAC, specifically intraductal papillary mucinous neoplasms (IPMNs), represent a
pathway to invasive ...neoplasia, although the molecular correlates of progression remain to be fully elucidated. Single-cell transcriptomics provides a unique avenue for dissecting both the epithelial and microenvironmental heterogeneities that accompany multistep progression from noninvasive IPMNs to PDAC.
Single-cell RNA sequencing was performed through droplet-based sequencing on 5,403 cells from 2 low-grade IPMNs (LGD-IPMNs), 2 high-grade IPMNs (HGD-IPMN), and 2 PDACs (all surgically resected).
Analysis of single-cell transcriptomes revealed heterogeneous alterations within the epithelium and the tumor microenvironment during the progression of noninvasive dysplasia to invasive cancer. Although HGD-IPMNs expressed many core signaling pathways described in PDAC, LGD-IPMNs harbored subsets of single cells with a transcriptomic profile that overlapped with invasive cancer. Notably, a proinflammatory immune component was readily seen in low-grade IPMNs, composed of cytotoxic T cells, activated T-helper cells, and dendritic cells, which was progressively depleted during neoplastic progression, accompanied by infiltration of myeloid-derived suppressor cells. Finally, stromal myofibroblast populations were heterogeneous and acquired a previously described tumor-promoting and immune-evading phenotype during invasive carcinogenesis.
This study demonstrates the ability to perform high-resolution profiling of the transcriptomic changes that occur during multistep progression of cystic PDAC precursors to cancer. Notably, single-cell analysis provides an unparalleled insight into both the epithelial and microenvironmental heterogeneities that accompany early cancer pathogenesis and might be a useful substrate to identify targets for cancer interception.
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Cancer cells release extracellular vesicles known as extracellular vesicles (EVs), containing tumor-derived DNA, RNA and proteins within their cargo, into the circulation. Circulating tumor-derived ...extracellular vesicles (TEV) can be used in the context of serial “liquid biopsies” for early detection of cancer, for monitoring disease burden in patients, and for assessing recurrence in the post-resection setting. Nonetheless, isolating sufficient TEV by ultracentrifugation-based approaches, in order to enable molecular assessment of EVs cargo, can be an arduous, time-consuming process and is inconsistent in the context of yield and purity among institutions. Herein, we describe a microfluidic platform, which we have named MITEV (Microfluidic Isolation of Tumor-derived Extracellular Vesicles) for the rapid isolation of TEV from the plasma of pancreatic cancer patients. The device, which has ~100,000 pillars placed in a zigzag pattern and is coated with antibodies against generic EV surface proteins (anti-CD63, -CD9, and -CD81 antibodies) or the TEV specific anti-Epithelial Cell Adhesion Molecule (EpCAM) antibody, is capable of high-throughput EVs isolation and yields sufficient DNA (total of ~2–14 ng from 2-ml plasma) for downstream genomic analysis. Using two independent quantitative platforms, droplet digital polymerase chain reaction (ddPCR) and molecular barcoding using nanoString nCounter® technology, we can reliably identify
KRAS
mutations within isolated TEV of treatment-naïve metastatic pancreatic cancer patients. Our study suggests that the MITEV device can be used for point-of-care applications, such as in the context of monitoring residual or recurrent tumor presence in pancreatic cancer patients undergoing therapy.
Circulating tumor cells (CTCs) shed from the primary tumor undergo significant fragmentation in the microvasculature, and very few escape to instigate metastases. Inspired by this in vivo behavior of ...CTCs, we report a microfluidic method to phenotype cancer cells based on their ability to arrest and fragment at a micropillar-based bifurcation. We find that in addition to cancer cell size, mechanical properties determine fragmentability. We observe that highly metastatic prostate cancer cells are more resistant to fragmentation than weakly metastatic cells, providing the first indication that metastatic CTCs can escape rupture and potentially initiate secondary tumors. Our method may thus be useful in identifying phenotypes that succumb to or escape mechanical trauma in microcirculation.
Introduction
Cell proliferation represents a major hallmark of cancer biology, and manifests itself in the assessment of tumor growth, drug resistance and metastasis. Tracking cell proliferation or ...cell fate at the single-cell level can reveal phenotypic heterogeneity. However, characterization of cell proliferation is typically done in bulk assays which does not inform on cells that can proliferate under given environmental perturbations. Thus, there is a need for single-cell approaches that allow longitudinal tracking of the fate of a large number of individual cells to reveal diverse phenotypes.
Methods
We fabricated a new microfluidic architecture for high efficiency capture of single tumor cells, with the capacity to monitor cell divisions across multiple daughter cells. This single-cell proliferation (SCP) device enabled the quantification of the fate of more than 1000 individual cancer cells longitudinally, allowing comprehensive profiling of the phenotypic heterogeneity that would be otherwise masked in standard cell proliferation assays. We characterized the efficiency of single cell capture and demonstrated the utility of the SCP device by exposing MCF-7 breast tumor cells to different doses of the chemotherapeutic agent doxorubicin.
Results
The single cell trapping efficiency of the SCP device was found to be ~ 85%. At the low doses of doxorubicin (0.01 µM, 0.001 µM, 0.0001 µM), we observed that 50–80% of the drug-treated cells had undergone proliferation, and less than 10% of the cells do not proliferate. Additionally, we demonstrated the potential of the SCP device in circulating tumor cell applications where minimizing target cell loss is critical. We showed selective capture of breast tumor cells from a binary mixture of cells (tumor cells and white blood cells) that was isolated from blood processing. We successfully characterized the proliferation statistics of these captured cells despite their extremely low counts in the original binary suspension.
Conclusions
The SCP device has significant potential for cancer research with the ability to quantify proliferation statistics of individual tumor cells, opening new avenues of investigation ranging from evaluating drug resistance of anti-cancer compounds to monitoring the replicative potential of patient-derived cells.
Abstract only
Survival of circulating tumor cells (CTCs) in blood circulation is essential for cancer metastasis. However, fluid forces in microcirculation have been shown to cause cell rupture, ...lending to the notion that such mechanical trauma of CTCs is responsible for metastatic inefficiency. Literature evidence for tumor cell death due to mechanical damage remains ambiguous and moreover the physical mechanisms of cell rupture are poorly understood. Here, we develop a microfluidic capillary model of microcirculation that contains an array of flow bifurcations and investigate rupture of tumor cells. We find that when a cancer cell arrives at a narrow bifurcation, it is temporarily arrested, followed by strong cellular deformation causing a cytosol‐filled membrane‐bound vesicle to be pinched‐off. The vesicle size was found to increase with the size of tumor cell suggesting that the cytosol content is larger for bigger cells. To identify the essential features of the mechanism underlying the rupture process, we used a library of drugs that (i) either enhance or inhibit the polymerization of actin and microtubules (ii) inhibit myosin II and decrease cortical tension, and (iii) condense and decondense nuclear chromatin. Results from these studies show that the cytoskeletal elements and membrane play an important role in regulating the time‐scale for rupture of tumor cells, but not the nucleus. The vesicle size was not sensitive to any of the drug treatments indicating again that the cytosol content determines the vesicle volume. Collectively, the drug studies support the poroelastic model of a cell, where under mechanical stress, the cytosol flows through the elastic cytoskeleton and fills the membrane that is detached from the cortex. Experiments with different cancer cell lines showed that highly metastatic cells take longer to rupture suggesting that their cytoskeleton and membrane properties are different than weakly metastatic cell lines. Finally, we find that less than 5% of the fragmented tumor cells undergo apoptosis, suggesting that CTCs can survive the impact of fluid forces in microcirculation and that mechanical trauma is not a significant cause for metastatic inefficiency.
Support or Funding Information
Cancer Prevention and Research Institute of Texas (Grant No. RP 140298)
Liquid biopsies in pancreatic cancer Kamyabi, Nabiollah; Bernard, Vincent; Maitra, Anirban
Expert review of anticancer therapy,
10/2019, Letnik:
19, Številka:
10
Journal Article
Recenzirano
Odprti dostop
: Pancreatic ductal adenocarcinoma (PDAC) is a disease of high lethality. Invasive tissue biopsies of primary or metastatic lesions remain the gold standard for diagnosis, but repeated sampling is ...infeasible. Noninvasive liquid biopsies offer new opportunities for early diagnosis for high-risk cohorts, and for the longitudinal analysis of tumor evolution and progression in patients on therapy. Liquid biopsies can capture tumor-associated components, such as circulating tumor DNA (ctDNA), extracellular vesicles (EVs), and circulating tumor cells (CTCs), each of which provides genomic and molecular information about the underlying PDAC that can potentially inform clinical decisions.
: Here, we reviewed current knowledge and recent technological advances regarding liquid biopsy in PDAC and mention the pitfalls and benefits in each methodology. We also discuss clinical correlative studies for diagnosis and prognosis in PDAC.
:In pancreatic cancer where tissue samples are limited and repeated tissue biopsies are mostly invasive and infeasible, liquid biopsies opened a new window for tumor diagnosis, molecular stratification, and treatment monitoring. While none of the isolation and analysis methods have gained widespread clinical acceptance, it is imperative that the advantages and limitations of each platform for isolation and analysis of tumor associated components are taken into consideration.
We aimed to investigate the clinical utility of circulating tumor cell DNA (ctDNA) and exosome DNA (exoDNA) in pancreatic cancer.
We collected liquid biopsy samples from 194 patients undergoing ...treatment for localized or metastatic pancreatic adenocarcinoma from April 7, 2015, through October 13, 2017 (425 blood samples collected before baseline and during therapy). Additional liquid biopsy samples were collected from 37 disease control individuals. Droplet digital polymerase chain reaction was used to determine KRAS mutant allele fraction (MAF) from ctDNA and exoDNA purified from plasma. For the longitudinal analysis, we analyzed exoDNA and ctDNA in 123 serial blood samples from 34 patients. We performed analysis including Cox regression, Fisher exact test, and Bayesian inference to associate KRAS MAFs in exoDNA and ctDNA with prognostic and predictive outcomes.
In the 34 patients with potentially resectable tumors, an increase in exoDNA level after neoadjuvant therapy was significantly associated with disease progression (P = .003), whereas ctDNA did not show correlations with outcomes. Concordance rates of KRAS mutations present in surgically resected tissue and detected in liquid biopsy samples were greater than 95%. On univariate analysis, patients with metastases and detectable ctDNA at baseline status had significantly shorter times of progression-free survival (PFS) (hazard ratio HR for death, 1.8; 95% CI, 1.1–3.0; P = .019), and overall survival (OS) (HR, 2.8; 95% CI, 1.4–5.7; P = .0045) compared with patients without detectable ctDNA. On multivariate analysis, MAFs ≥5% in exoDNA were a significant predictor of PFS (HR, 2.28; 95% CI, 1.18–4.40; P = .014) and OS (HR, 3.46; 95% CI, 1.40–8.50; P = .007). A multianalyte approach showed detection of both ctDNA and exoDNA MAFs ≥5% at baseline status to be a significant predictor of OS (HR, 7.73, 95% CI, 2.61–22.91, P = .00002) on multivariate analysis. In the longitudinal analysis, an MAF peak above 1% in exoDNA was significantly associated with radiologic progression (P = .0003).
In a prospective cohort of pancreatic cancer patients, we show how longitudinal monitoring using liquid biopsy samples through exoDNA and ctDNA provides both predictive and prognostic information relevant to therapeutic stratification.
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Limited accessibility of the tumour precludes longitudinal characterisation for therapy guidance in pancreatic ductal adenocarcinoma (PDAC).
We utilised dielectrophoresis-field flow fractionation ...(DEP-FFF) to isolate circulating tumour cells (CTCs) in 272 blood draws from 74 PDAC patients (41 localised, 33 metastatic) to non-invasively monitor disease progression.
Analysis using multiplex imaging flow cytometry revealed four distinct sub-populations of CTCs: epithelial (E-CTC), mesenchymal (M-CTC), partial epithelial-mesenchymal transition (pEMT-CTC) and stem cell-like (SC-CTC). Overall, CTC detection rate was 76.8% (209/272 draws) and total CTC counts did not correlate with any clinicopathological variables. However, the proportion of pEMT-CTCs (prop-pEMT) was correlated with advanced disease, worse progression-free and overall survival in all patients, and earlier recurrence after resection.
Our results underscore the importance of immunophenotyping and quantifying specific CTC sub-populations in PDAC.
Human intestinal enteroids (HIE) models have contributed significantly to our understanding of diarrheal diseases and other intestinal infections, but their routine culture conditions fail to mimic ...the mechanical environment of the native intestinal wall. Because the mechanical characteristics of the intestine significantly alter how pathogens interact with the intestinal epithelium, we used different concentrations of polyethylene glycol (PEG) to generate soft (~2 kPa), medium (~10 kPa), and stiff (~100 kPa) hydrogel biomaterial scaffolds. The height of HIEs cultured in monolayers atop these hydrogels was 18 µm whereas HIEs grown on rigid tissue culture surfaces (with stiffness in the GPa range) were 10 µm. Substrate stiffness also influenced the amount of enteroaggregative E. coli (EAEC strain 042) adhered to the HIEs. We quantified a striking difference in adherence pattern; on the medium and soft gels, the bacteria formed clusters of > 100 and even > 1000 on both duodenal and jejunal HIEs (such as would be found in biofilms), but did not on glass slides and stiff hydrogels. All hydrogel cultured HIEs showed significant enrichment for gene and signaling pathways related to epithelial differentiation, cell junctions and adhesions, extracellular matrix, mucins, and cell signaling compared to the HIEs cultured on rigid tissue culture surfaces. Collectively, these results indicate that the HIE monolayers cultured on the hydrogels are primed for a robust engagement with their mechanical environment, and that the soft hydrogels promote the formation of larger EAEC aggregates, likely through an indirect differential effect on mucus.
Enteroids are a form of in vitro experimental mini-guts created from intestinal stem cells. Enteroids are usually cultured in 3D within Matrigel atop rigid glass or plastic substrates, which fail to mimic the native intestinal mechanical environment. Because intestinal mechanics significantly alter how pathogens interact with the intestinal epithelium, we grew human intestinal enteroids in 2D atop polyethylene glycol (PEG) hydrogel scaffolds that were soft, medium, or stiff. Compared with enteroids grown in 2D atop glass or plastic, the enteroids grown on hydrogels were taller and more enriched in mechanobiology-related gene signaling pathways. Additionally, enteroids on the softest hydrogels supported adhesion of large aggregates of enteroaggregative E. coli. Thus, this platform offers a more biomimetic model for studying enteric diseases.
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Human intestinal epithelial organoids (enteroids and colonoids) are tissue cultures used for understanding the physiology of the human intestinal epithelium. Here, we explored the effect on the ...transcriptome of common variations in culture methods, including extracellular matrix substrate, format, tissue segment, differentiation status, and patient heterogeneity. RNA-sequencing datasets from 276 experiments performed on 37 human enteroid and colonoid lines from 29 patients were aggregated from several groups in the Texas Medical Center. DESeq2 and gene set enrichment analysis (GSEA) were used to identify differentially expressed genes and enriched pathways. PERMANOVA, Pearson's correlation, and dendrogram analysis of the data originally indicated three tiers of influence of culture methods on transcriptomic variation: substrate (collagen vs. Matrigel) and format (3-D, transwell, and monolayer) had the largest effect; segment of origin (duodenum, jejunum, ileum, colon) and differentiation status had a moderate effect; and patient heterogeneity and specific experimental manipulations (e.g., pathogen infection) had the smallest effect. GSEA identified hundreds of pathways that varied between culture methods, such as IL1 cytokine signaling enriched in transwell versus monolayer cultures and E2F target genes enriched in collagen versus Matrigel cultures. The transcriptional influence of the format was furthermore validated in a synchronized experiment performed with various format-substrate combinations. Surprisingly, large differences in organoid transcriptome were driven by variations in culture methods such as format, whereas experimental manipulations such as infection had modest effects. These results show that common variations in culture conditions can have large effects on intestinal organoids and should be accounted for when designing experiments and comparing results between laboratories. Our data constitute the largest RNA-seq dataset interrogating human intestinal epithelial organoids.
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