Jianyu Rao

Nanomechanical analysis of cells from cancer patients

Change in cell stiffness is a new characteristic of cancer cells that affects the way they spread. Despite several studies on architectural changes in cultured cell lines, no ex vivo mechanical analyses of cancer cells obtained from patients have been reported. Using atomic force microscopy, we report the stiffness of live metastatic cancer cells taken from the body (pleural) fluids of patients with suspected lung, breast and pancreas cancer. Within the same sample, we find that the cell stiffness of metastatic cancer cells is more than 70% softer, with a standard deviation over five times narrower, than the benign cells that line the body cavity. Different cancer types were found to display a common stiffness. Our work shows that mechanical analysis can distinguish cancerous cells from normal ones even when they show similar shapes. These results show that nanomechanical analysis correlates well with immunohistochemical testing currently used for detecting cancer.

Hydrodynamic stretching of single cells for large population mechanical phenotyping

Cell state is often assayed through measurement of biochemical and biophysical markers. Although biochemical markers have been widely used, intrinsic biophysical markers, such as the ability to mechanically deform under a load, are advantageous in that they do not require costly labeling or sample preparation. However, current techniques that assay cell mechanical properties have had limited adoption in clinical and cell biology research applications. Here, we demonstrate an automated microfluidic technology capable of probing single-cell deformability at approximately 2,000 cells/s. The method uses inertial focusing to uniformly deliver cells to a stretching extensional flow where cells are deformed at high strain rates, imaged with a high-speed camera, and computationally analyzed to extract quantitative parameters. This approach allows us to analyze cells at throughputs orders of magnitude faster than previously reported biophysical flow cytometers and single-cell mechanics tools, while creating easily observable larger strains and limiting user time commitment and bias through automation. Using this approach we rapidly assay the deformability of native populations of leukocytes and malignant cells in pleural effusions and accurately predict disease state in patients with cancer and immune activation with a sensitivity of 91% and a specificity of 86%. As a tool for biological research, we show the deformability we measure is an early biomarker for pluripotent stem cell differentiation and is likely linked to nuclear structural changes. Microfluidic deformability cytometry brings the statistical accuracy of traditional flow cytometric techniques to label-free biophysical biomarkers, enabling applications in clinical diagnostics, stem cell characterization, and single-cell biophysics.

Green tea extract and (−)-epigallocatechin-3-gallate inhibit hypoxia- and serum-induced HIF-1α protein accumulation and VEGF expression in human cervical carcinoma and hepatoma cells

Green tea extract and its major component (−)-epigallocatechin-3-gallate (EGCG) exhibit antiangiogenic activities in various experimental tumor models. A growing body of evidence has established that hypoxia-inducible factor-1α (HIF-1α) and its downstream target, vascular endothelial growth factor (VEGF), play a critical role in tumor angiogenesis. In this study, we investigated the effect of green tea extract and EGCG on HIF-1α and VEGF expression in human cervical carcinoma (HeLa) and hepatoma (HepG2) cells. Our results showed that green tea extract and EGCG significantly inhibited hypoxia- and serum-induced HIF-1α protein accumulation in these cancer cells but had no effects on HIF-1α mRNA expression. Suppression of HIF-1α protein by green tea extract and EGCG also resulted in a drastic decrease in VEGF expression at both mRNA and protein levels. The mechanisms of green tea extract and EGCG inhibition of hypoxia-induced HIF-1α protein accumulation seem to involve the blocking of both phosphatidylinositol 3-kinase/Akt and extracellular signal-regulated kinase 1/2 signaling pathways and the enhancing of HIF-1α protein degradation through the proteasome system. In addition, green tea extract and EGCG inhibited serum-induced HIF-1α protein and VEGF expression by interfering with the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin signaling pathways, which play a crucial role in the protein translational machinery cascade. Functionally, green tea extract and EGCG abolished both chemoattractant- and hypoxia-stimulated HeLa cell migration. Our data suggested that HIF-1α/VEGF function as therapeutic target for green tea extract and EGCG in the context of cancer chemoprevention and anticancer therapy. [Mol Cancer Ther 2006;5(5):1227–38]

Genome-wide association study in Chinese men identifies two new prostate cancer risk loci at 9q31.2 and 19q13.4

Prostate cancer risk–associated variants have been reported in populations of European descent, African-Americans and Japanese using genome-wide association studies (GWAS). To systematically investigate prostate cancer risk–associated variants in Chinese men, we performed the first GWAS in Han Chinese. In addition to confirming several associations reported in other ancestry groups, this study identified two new risk-associated loci for prostate cancer on chromosomes 9q31.2 (rs817826, P = 5.45 × 10−14) and 19q13.4 (rs103294, P = 5.34 × 10−16) in 4,484 prostate cancer cases and 8,934 controls. The rs103294 marker at 19q13.4 is in strong linkage equilibrium with a 6.7-kb germline deletion that removes the first six of seven exons in LILRA3, a gene regulating inflammatory response, and was significantly associated with the mRNA expression of LILRA3 in T cells (P < 1 × 10−4). These findings may advance the understanding of genetic susceptibility to prostate cancer.

Identification and characterization of repopulating spermatogonial stem cells from the adult human testis

BACKGROUND This study was conducted to identify and characterize repopulating spermatogonial stem cells (SSCs) in the adult human testes. METHODS Testes biopsies from obstructive azoospermic patients and normal segments of human testicular tissue were used. Flow cytometry, real-time PCR and immunohistochemical analysis were performed. Purified human spermatogonia were transplanted into busulfan-treated recipient mouse testes and integrated cells were detected by human nuclear protein antibody co-localized with stem cell and germ cell markers. RESULTS Testicular biopsies collected from obstructive azoospermic men showed similar morphology and distribution of markers to the normal human testes. Flow cytometry showed distinct populations of stage-specific embryonic antigen-4 (SSEA-4), CD49f and CD90 positive cells in the adult human testes. SSEA-4 (+) cells showed high expression levels of SSC-specific genes and high levels of telomerase activity. Extensive colonization of human cells in the mouse testes indicates the presence of highly enriched populations of SSCs in the SSEA-4 (+) sorted cells. All the HNP (+) cells in the mouse testes were positive for germ cell marker dead box mRNA helicase and only half of them were dimly positive for c-kit. In addition, subpopulations of human spermatogonia that colonized mouse testes were positively stained for CD49f, GPR-125, Nanog and Oct-4 indicating the existence of population of cells among human spermatogonia with SSC and pluripotent characteristics. CONCLUSIONS This study clearly demonstrates that repopulating human SSCs have phenotypic characteristics of SSEA-4(+), CD49f(+), GPR-125(+)and c-Kit (neg/low). The results have direct implications for enrichment of human spermatogonia for further culture and germ cell differentiation studies.

Quantitative Diagnosis of Malignant Pleural Effusions by Single-Cell Mechanophenotyping

Single-cell biophysical properties were used for diagnosing malignant pleural effusions from patients. Cytometry Device Helps (De)form a Diagnosis Is it benign, or malignant? That is the main concern of cytopathologists as they screen cells in pleural effusions, taken from the lungs of patients suspected of having infections or cancer. This process is subjective and time-intensive and requires an expert’s eye. So, to quickly “prescreen” samples for malignancy (and follow-up), Tse et al. describe deformability cytometry (DC)—an approach that relies on microfluidic forces to diagnose pleural effusion samples as malignant, or not. The authors’ device accelerates effusion samples through two opposing microfluidic channels. At the channels’ four-way intersection, the cells are rapidly decelerated as they encounter the opposing flow, and then exit out the side channels. This leads to cell deformation, changing them from sphere-like shapes to pancakes. High-speed video of this intersection allowed Tse et al. to quantify cellular squishing: the more deformable the cell, the more malignant it is. The authors took 119 pleural effusion samples from patients with known clinical outcomes—negative for malignant cells (benign), acute inflammation, chronic/mixed inflammation, atypical cells, and malignant pleural effusions (MPEs)—to develop a diagnostic scoring system on a scale of 1 to 10, with 1 being benign. DC showed the best predictive abilities in two high-confidence regimes: 1 to 6 and 9 to 10. Scores of 7 and 8 were more difficult to diagnose, so these may be the types of samples where a cytopathologist’s initial input would be necessary. Importantly, the authors looked at samples from patients that were cytology-negative with concurrent malignancy, such as a tumor, but 6 months later were diagnosed with disseminated disease. Five of 10 patients with high-grade cancers that were cytology-negative at sample collection scored high using DC. This suggests that the DC tool could be used to screen early for MPE. Using deformability as a marker of disease will require additional validation in pleural effusion samples from patients with many different types of cancer. Nevertheless, owing to the ease of use and objective readout, with further clinical testing, DC should be useful as a quick screening tool to form an early diagnosis of MPEs. Biophysical characteristics of cells are attractive as potential diagnostic markers for cancer. Transformation of cell state or phenotype and the accompanying epigenetic, nuclear, and cytoplasmic modifications lead to measureable changes in cellular architecture. We recently introduced a technique called deformability cytometry (DC) that enables rapid mechanophenotyping of single cells in suspension at rates of 1000 cells/s—a throughput that is comparable to traditional flow cytometry. We applied this technique to diagnose malignant pleural effusions, in which disseminated tumor cells can be difficult to accurately identify by traditional cytology. An algorithmic diagnostic scoring system was developed on the basis of quantitative features of two-dimensional distributions of single-cell mechanophenotypes from 119 samples. The DC scoring system classified 63% of the samples into two high-confidence regimes with 100% positive predictive value or 100% negative predictive value, and achieved an area under the curve of 0.86. This performance is suitable for a prescreening role to focus cytopathologist analysis time on a smaller fraction of difficult samples. Diagnosis of samples that present a challenge to cytology was also improved. Samples labeled as “atypical cells,” which require additional time and follow-up, were classified in high-confidence regimes in 8 of 15 cases. Further, 10 of 17 cytology-negative samples corresponding to patients with concurrent cancer were correctly classified as malignant or negative, in agreement with 6-month outcomes. This study lays the groundwork for broader validation of label-free quantitative biophysical markers for clinical diagnoses of cancer and inflammation, which could help to reduce laboratory workload and improve clinical decision-making.

Autocrine activation of PDGFRα promotes the progression of ovarian cancer

Platelet-derived growth factor receptor (PDGFR)α expression was found in ovarian cancer cells and tumors by microarray hybridization. This led us to test whether ovarian cancers also produce ligands for this receptor, as this would demonstrate that such malignancies support their own growth and spread through autocrine activation. We assayed the expression of ligands for the PDGFR in ovarian tumors, cell lines and peritoneal fluid using RT–PCR, immunohistochemistry (IHC) and ELISA. We detected strong mRNA expression for the PDGFRα ligands in most ovarian tumors. Receptor and ligand expressions (PDGFRα and PDGF AB) were also detected by IHC in, respectively, 34 and 32 of 47 ovarian tumors. The stainings for PDGFRα and PDGF AB were strongly correlated (P-value=0.014), suggesting that an autocrine loop is functional in ovarian cancer. PDGF AA and BB were quantified in peritoneal fluid by ELISA. Both ligands are secreted at higher levels in ovarian cancer ascites specimens (n=54) than in fluid from nonmalignant disorders (n=8). PDGF was detected in media conditioned by ovarian cancer cells. Such conditioned media induced activation of the PDGFR, Akt and MAPK and stimulated cell proliferation. A neutralizing PDGF antibody blocked these effects. Specific PDGFR inhibition by siRNA or a neutralizing antibody to the receptor inhibited PDGF-stimulated receptor activation and cell proliferation, suggesting that receptor targeting has a role in ovarian cancer treatment.

Green tea drinking and multigenetic index on the risk of stomach cancer in a Chinese population

The purpose of our study was to examine the roles of green tea drinking, other risk and protective factors, and polymorphism of susceptibility genes such as GSTM1, GSTT1, GSTP1, and p53 codon 72 and their possible joint effects on the risk of stomach cancer. A population‐based case‐control study was conducted in Taixing, China, including 206 newly diagnosed cases with stomach cancer and 415 healthy control subjects. Epidemiological data were collected by in‐person interviews using a standard questionnaire. Polymorphisms of susceptibility genes were assayed by PCR‐RFLP techniques. A multigenetic index was created by summing up the number of risk genotypes. The data were analyzed using the logistic regression model. A reverse association between green tea drinking and risk of stomach cancer was observed with an adjusted odds ratio (OR) of 0.59 (95% confidence interval [CI] = 0.34–1.01). Dose‐response relationship was shown (p‐trend < 0.05). A higher score on the multigenetic index was associated with increased risk of stomach cancer with an adjusted OR of 2.21 (95% CI = 1.02–4.79) for those with at least 3 risk genotypes compared to those with <2 risk genotypes. Green tea drinking was suggested to have more than multiplicative interactions with alcohol consumption with an adjusted OR for interaction of 4.57 (95% CI = 1.62–12.89), and with higher multigenetic index with adjusted OR for interaction of 2.31 (95% CI = 0.88–6.03). The protective effect of green tea drinking was observed on the risk of stomach cancer and the possible effect modification by susceptibility genes was suggested.

Gene expression in epithelial ovarian carcinoma

We analysed the mRNA levels corresponding to 12 600 transcripts in primary cultures of ovarian epithelial cells derived from nine normal ovaries and 21 epithelial ovarian carcinoma. The class distinction and hierarchical clustering of expression data revealed a clear distinction in gene expression between normal and carcinoma-derived ovarian epithelial cells. Comparison of expression levels revealed 111 genes with mean expression values of >2.5-fold higher in carcinoma cells. Similarly, 62 genes were expressed at >2.5-fold higher levels in normal ovarian epithelial cells. For a few selected genes, we demonstrate that the pattern of differential expression observed in cultured epithelial cells is present in the normal ovaries and epithelial ovarian carcinoma. Use of cultured epithelial cells represents a novel strategy to study gene expression in a cell-type specific manner.

Comparison of methods for DNA extraction from paraffin-embedded tissues and buccal cells

Both paraffin-embedded tissue specimens and buccal cells are excellent resources for large-scale molecular epidemiological studies. In order to identify the optimal method for DNA extraction, we compared three methods: (1) modified phenol-chloroform protocol; (2) simple boiling method; and (3) DNA Extraction Mini Kit. For paraffin-embedded tissue specimens, amplification of the beta-globin gene sequence was successful in 30 of 34 (88.2%) by the simple boiling method, 29 of 34 (85.3%) samples using DNA extracted by the phenol-chloroform method, and 18 of 34 (52.9%) by the DNA Mini Kit. For buccal cells, amplification of the beta-globin gene sequence was successful in 16 of 17 (94.1%) DNA samples extracted by the phenol-chloroform method, 2 of 16 (12.5%) by the simple boiling method, and 12 of 16 (75%) by the DNA Mini Kit. Both the simple boiling method and the phenol-chloroform method are better methods for DNA isolation from paraffin-embedded tissue specimens, and the phenol-chloroform method is the best method for DNA extraction from buccal cells.