Cancer Tissue Sections Research Articles

Abstract 2106: Focal gene expression profiling of counterstained FFPE with correlation to morphology using TempO-Seq

Abstract We previously reported on use of the whole transcriptome TempO-Seq® assay to profile gene expression from 1 mm2 focal areas of 5 µm thick FFPE sections of normal and cancerous tissue to identify disease biomarkers and mechanistic pathways. We are now presenting data demonstrating that the TempO-Seq assay can be performed as an “in situ” assay on slides by an automated slide stainer, followed by counterstaining. Then, using an automated digital imaging platform, areas as small as 30 μm in diameter within the FFPE section can be profiled, permitting the gene expression data to be correlated directly to the specific morphology of that focal area. The pathologist uses a computer interface to select areas for profiling during the course of the histologic examination of the section, and then the TempO-Seq probes are automatically recovered from those region(s) of interest. After depositing them into PCR tubes, the assay is completed with PCR amplification and sample barcoding before pooling the samples into a library for sequencing. Analysis of the sequencing data is carried out automatically to report results. There is no use of laser capture or destruction of the tissue. The TempO-Seq processed slides can be archived and additional areas may be sampled at a later date. In this study, we sample replicate areas of matched normal vs. cancerous tissue, measuring gene biomarkers of clinical utility, gene fusions, and SNPs. We present gene expression profiles for replicate areas of pure stroma, single normal glands, single high-grade PIN glands, and single cancerous glands from prostate FFPE, demonstrating the statistical power of the TempO-Seq in situ assay as well as the correlation to focal histology and the use of focal differential expression to elucidate molecular pathways involved in the transition from normal tissue to cancer. We report measurements of clinically relevant biomarkers and signatures from clinical FFPE samples, plus therapeutic targets and drug resistance genes. This new approach demonstrates that complex molecular tests can be carried out by any pathologist in their own lab, and makes moot the issues of “% cancer” and amount of tissue required for testing. The approach we present brings extraction-free complex molecular testing of FFPE into the pathology lab and takes molecular pathology to a new level of simplicity, focal precision and correlation to morphology. Citation Format: Elliot Imler, Milos Babic, Deanna Adams, Peter Shepard, Joanne Yeakley, Raymond B. Nagle, Bruce Seligmann. Focal gene expression profiling of counterstained FFPE with correlation to morphology using TempO-Seq [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 2106.

PO-463 Multiparametric analysis of lung cancer tissue sections using imaging mass cytometry

IntroductionA novel tool for multiparametric molecular pathology analysis of tissue sections is described. Imaging Mass Cytometry (IMC), a new mass spectrometer-based technology, provides visualisation and spatial distribution of up to 40 markers in a single tissue section with 1 µm resolution. IMC uses metal-tagged antibodies (mtAbs), a pulsed UV laser for ablating regions of interest, and a stream of inert gas to transfer material generated by the laser to the mass cytometer.1Material and methodsTissue microarray (TMA) of human lung cancer was stained according to IMC protocol2 using a mix of 23 mtAbs.The samples were inserted into the ablation chamber of the Hyperion Imaging System. CyTOF® Software v6.7 and MCD Viewer 1.0 were used for data acquisition and rendering of pseudo three-colour images. Quantification of selected markers was done with Definiens Tissue Studio® (DTS) software. Standard HPA protocol was used for immunohistochemical (IHC) staining.Results and discussionsIMC analysis of human lung cancer TMA stained with a panel of 23 mtAbs revealed that core morphology was consistent with anatomopathological tumour classification. Immune cell phenotype and spatial distribution assessed by IHC and IMC were equivalent.IMC generated data was used to quantitate PD-L1 marker expression. Percentages of cells expressing different levels of PD-L1 and average intensity of PD-L1 related signal were calculated for tumour and stroma regions of each core. In addition, PD-L1 corresponding signal intensity was calculated in cytoplasm and membrane compartments of marker-positive cells.ConclusionIMC technology is a powerful tool for multiparametric histopathological analysis. It allows researchers to simultaneously detect up to 40 markers with single-cell detection resolution equivalent to conventional IHC. Using DTS software we demonstrate quantitation of multiple markers in lung tumour sections probed with a panel of 23 mtAbs.ReferencesChang Q, Ornatsky OI, Siddiqui I, Loboda A, Baranov VI, Hedley DW. Imaging Mass Cytometry. Cytometry A 2017;91:160–169.Chang Q, Ornatsky O, Hedley D. Staining of frozen and formalin-fixed, paraffin-embedded tissues with metal-labeled antibodies for Imaging Mass Cytometry analysis. Curr Protoc Cytom, 2017;82(October):12.47.1–12.47.8.

Abstract 5651: Achieving simultaneous improvements in workflow and multiplexing in whole-slide tissue imaging

Abstract With very high spatial and biological complexity, tumor micro-environments present a significant characterization challenge, which is only heightened by the need to image entire sections of cancer tissue on many different samples. A variety of techniques offer very high levels of multiplexing, but always fail on either speed requirements (incredibly long times required to image a whole slide) or on compatibility issues (expensive, hard-to-operate CapEx not suitable for high-throughput CLIA operation). Ultivue has developed a platform reagent technology (InSituPlex™) that simultaneously enables whole- slide higher multiplexing by immunofluorescence AND complete compatibility with existing instrumentation. The InSituPlex technology comprises five key foundational elements. First, short unique DNA barcodes are covalently attached to each primary antibody (Ab). These conjugates are purified and functionally characterized in tissue for optimal staining specificity. Importantly, all of this work is done in advance by Ultivue. Second, tissue samples are stained in a single step. Third, all the DNA sequences attached to targets (via primary Ab binding) are simultaneously amplified, allowing for detection of less abundant markers e.g. PD-1, PDL-1 etc. Fourth, complementary DNA sequences each with different, covalently- attached fluorescent labels (“imager strands”) are simultaneously hybridized to the amplified DNA on the targets. All these wet chemistry steps are carried out in walkaway mode using commercially available auto-stainers. The samples are then imaged using a standard, whole-slide multi-color fluorescence imaging system. Finally, in an optional fifth step, the imager strands are gently removed via an enzymatic dehybridzation- with complete retention of antigenicity- to enable higher multiplexing via additional rounds of hybridization/imaging. The InSituPlex reagent technology offers many advantages for cancer research over current approaches: • the ability to detect 5, 10, or more targets simultaneously via whole-slide imaging on an each and every sample, with no loss in antigenicity; • the ability to leverage existing auto-stainers and existing fluorescent microscopes with Ultivue's reagent kits to carry out high-throughput analyses of multiple cancer biomarkers; and • the ability to design custom biomarker panels that leverage Ultivue's unique barcoding capability and infrastructure. This poster will elaborate on each step of the technology, and provide multiple examples of data acquired using different biomarker panels and different cancer tissue types. Citation Format: Michael Natan. Achieving simultaneous improvements in workflow and multiplexing in whole-slide tissue imaging [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 5651.

Elevated GPC3 level promotes cell proliferation in liver cancer.

The aim of the present study was to investigate the biological role of glypican 3 (GPC3), and to identify its mechanism and clinical significance in the carcinogenesis of liver cancer. A total of 114 patients with liver cancer were involved. Their clinical data, hematoxylin and eosin-stained and Antigen Ki-67 protein (Ki-67) and GPC3 immunohistochemically-stained liver cancer tissue sections were analyzed to evaluate the correlation between the liver cancer proliferation, differentiation and GPC3 expression. Fluorescence microscopy, western blotting, MTT and reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays were performed in HepG2 and HLE cell lines to investigate the potential mechanisms of action. Among the 114 patients with liver cancer enrolled in the present study, 12 exhibited well-differentiated liver cancer, of which 6 (50%) were positive for GPC3. A total of 30 cases exhibited poorly differentiated liver cancer; 26 (87%) of these expressed GPC3 and 11 cases (37%) demonstrated strong positive expression levels. The other 72 liver cancer cases were moderately differentiated; 75% (54/72) of these expressed GPC3 and 12.5% (9/72) exhibited strong positive expression levels. There was a significant association between the levels of GPC3 expression and liver cancer differentiation (χ2=16.306, P=0.008). Ki-67 staining as the criteria of the liver cancer cell proliferation index also indicated a cross correlation between liver cancer proliferation and GPC3 levels. Among the 39 liver cancer samples with a cell proliferation index <5%, only 2.6% (1/39) exhibited strong positive GPC3 staining, but of the 16 cases with a high cell proliferation index >50%, 6 exhibited strong GPC3 staining (37.5%). The difference of cell proliferation indexes between cancer cells were well, moderate and poorly differentiated, and was markedly significant (χ2=26.334, P=0.002), and suggested that liver cancer cell proliferation was positively correlated with GPC3 expression (r=0.316, P=0.001). Consistently, in vitro analysis indicated that GPC3 promoted HepG2 and HLE cell growth, which was more apparent in HepG2 cells. The RT-qPCR results indicated that GPC3 promoted proliferation through the Hedgehog (Hh) pathway in HepG2 cells, but not in HLE cells. In the present study, it was demonstrated that patients with liver cancer with higher GPC3 levels exhibited poorer differentiation and higher proliferation levels. In vitro GPC3 may promote liver cancer cell lines proliferation through the Hh pathway.

Knockdown of Ran GTPase expression inhibits the proliferation and migration of breast cancer cells.

Breast cancer is the second leading cause of cancer-associated mortality in women worldwide. Strong evidence has suggested that Ran, which is a small GTP binding protein involved in the transport of RNA and protein across the nucleus, may be a key cellular protein involved in the metastatic progression of cancer. The present study investigated Ran gene expression in breast cancer tissue samples obtained from 140 patients who had undergone surgical resection for breast cancer. Western blot analysis of Ran in breast cancer tissues and paired adjacent normal tissues showed that expression of Ran was significantly increased in breast cancer tissues. Immunohistochemistry analyses conducted on formalin-fixed paraffin-embedded breast cancer tissue sections revealed that Ran expression was associated with tumor histological grade, nerve invasion and metastasis, vascular metastasis and Ki-67 expression (a marker of cell proliferation). Kaplan-Meier survival analysis showed that increased Ran expression in patients with breast cancer was positively associated with a poor survival prognosis. Furthermore, in vitro experiments demonstrated that highly migratory MDA-MB-231 cancer cells treated with Ran-si-RNA (si-Ran), which knocked down expression of Ran, exhibited decreased motility in trans-well migration and wound healing assays. Cell cycle analysis of Ran knocked down MDA-MB-231 cells implicated Ran in cell cycle arrest and the inhibition of proliferation. Furthermore, a starvation and re-feeding (CCK-8) assay was performed, which indicated that Ran regulated breast cancer cell proliferation. Taken together, the results provide strong in vitro evidence of the involvement of Ran in the progression of breast cancer and suggest that it could have high potential as a therapeutic target and/or marker of disease.

Elevated EGF Levels in the Blood Serum of Dogs with Periodontal Diseases and Oral Tumours.

Paradontopathy and neoplasms of the oral cavity represent one of the greatest challenges in human and animal dentistry. EGF plays a key role in maintaining the integrity and proper rate of cell proliferation in normal oral epithelium. The aim of the present study was to study serum levels of EGF in dogs diagnosed with periodontal diseases and oral cavity tumours. The samples comprised of cancerous tissue sections and serum obtained from dogs of various breeds, aged between 5-13 years. Serum EGF concentrations were measured by an immunoenzymatic method. The median for EGF concentration in serum of dogs suffered from severe periodontal diseases was greater when compared to the control group. EGF concentration in dogs with malignant tumours was significantly higher than in those with non-malignant growths. A positive correlation between EGF concentration and tumour size was also observed. EGF level in dogs diagnosed with benign tumours was comparable to the control group. The blood serum level of EGF increases significantly in patients with malignant oral tumours and advanced periodontal disease. In malignant tumours, the high level of EGF correlates with the size and invasiveness of the neoplasm.

Spatial-Resolution Cell Type Proteome Profiling of Cancer Tissue by Fully Integrated Proteomics Technology.

Increasing attention has been focused on cell type proteome profiling for understanding the heterogeneous multicellular microenvironment in tissue samples. However, current cell type proteome profiling methods need large amounts of starting materials which preclude their application to clinical tumor specimens with limited access. Here, by seamlessly combining laser capture microdissection and integrated proteomics sample preparation technology SISPROT, specific cell types in tumor samples could be precisely dissected with single cell resolution and processed for high-sensitivity proteome profiling. Sample loss and contamination due to the multiple transfer steps are significantly reduced by the full integration and noncontact design. H&E staining dyes which are necessary for cell type investigation could be selectively removed by the unique two-stage design of the spintip device. This easy-to-use proteome profiling technology achieved high sensitivity with the identification of more than 500 proteins from only 0.1 mm2 and 10 μm thickness colon cancer tissue section. The first cell type proteome profiling of four cell types from one colon tumor and surrounding normal tissue, including cancer cells, enterocytes, lymphocytes, and smooth muscle cells, was obtained. 5271, 4691, 4876, and 2140 protein groups were identified, respectively, from tissue section of only 5 mm2 and 10 μm thickness. Furthermore, spatially resolved proteome distribution profiles of enterocytes, lymphocytes, and smooth muscle cells on the same tissue slices and across four consecutive sections with micrometer distance were successfully achieved. This fully integrated proteomics technology, termed LCM-SISPROT, is therefore promising for spatial-resolution cell type proteome profiling of tumor microenvironment with a minute amount of clinical starting materials.

Whole-Slide Mitosis Detection in H&E Breast Histology Using PHH3 as a Reference to Train Distilled Stain-Invariant Convolutional Networks.

Manual counting of mitotic tumor cells in tissue sections constitutes one of the strongest prognostic markers for breast cancer. This procedure, however, is time-consuming and error-prone. We developed a method to automatically detect mitotic figures in breast cancer tissue sections based on convolutional neural networks (CNNs). Application of CNNs to hematoxylin and eosin (H&E) stained histological tissue sections is hampered by noisy and expensive reference standards established by pathologists, lack of generalization due to staining variation across laboratories, and high computational requirements needed to process gigapixel whole-slide images (WSIs). In this paper, we present a method to train and evaluate CNNs to specifically solve these issues in the context of mitosis detection in breast cancer WSIs. First, by combining image analysis of mitotic activity in phosphohistone-H3 restained slides and registration, we built a reference standard for mitosis detection in entire H&E WSIs requiring minimal manual annotation effort. Second, we designed a data augmentation strategy that creates diverse and realistic H&E stain variations by modifying H&E color channels directly. Using it during training combined with network ensembling resulted in a stain invariant mitosis detector. Third, we applied knowledge distillation to reduce the computational requirements of the mitosis detection ensemble with a negligible loss of performance. The system was trained in a single-center cohort and evaluated in an independent multicenter cohort from the cancer genome atlas on the three tasks of the tumor proliferation assessment challenge. We obtained a performance within the top three best methods for most of the tasks of the challenge.

A simple model for cell type recognition using 2D-correlation analysis of FTIR images from breast cancer tissue

Breast cancer is the second most common cancer after lung cancer. So far, in clinical practice, most cancer parameters originating from histopathology rely on the visualization by a pathologist of microscopic structures observed in stained tissue sections, including immunohistochemistry markers. Fourier transform infrared spectroscopy (FTIR) spectroscopy provides a biochemical fingerprint of a biopsy sample and, together with advanced data analysis techniques, can accurately classify cell types. Yet, one of the challenges when dealing with FTIR imaging is the slow recording of the data. One cm2 tissue section requires several hours of image recording. We show in the present paper that 2D covariance analysis singles out only a few wavenumbers where both variance and covariance are large. Simple models could be built using 4 wavenumbers to identify the 4 main cell types present in breast cancer tissue sections. Decision trees provide particularly simple models to reach discrimination between the 4 cell types. The robustness of these simple decision-tree models were challenged with FTIR spectral data obtained using different recording conditions. One test set was recorded by transflection on tissue sections in the presence of paraffin while the training set was obtained on dewaxed tissue sections by transmission. Furthermore, the test set was collected with a different brand of FTIR microscope and a different pixel size. Despite the different recording conditions, separating extracellular matrix (ECM) from carcinoma spectra was 100% successful, underlying the robustness of this univariate model and the utility of covariance analysis for revealing efficient wavenumbers. We suggest that 2D covariance maps using the full spectral range could be most useful to select the interesting wavenumbers and achieve very fast data acquisition on quantum cascade laser infrared imaging microscopes.

Increased cytomegalovirus replication by 5-Azacytidine and viral-induced cytoplasmic expression of DNMT‑1 in medulloblastoma and endothelial cells.

Among all brain tumors diagnosed in children, medulloblastomas (MBs) are associated with a poor prognosis. The etiology of MB is not fully understood, yet the impact of epigenetic alterations of oncogenes has previously been established. During the past decade, the human cytomegalovirus (HCMV) has been detected in several types of cancer, including MB. Since DNA methylation occurs in the cell nucleus and this is considered a host defence response, we studied the impact of HCMV infection on DNA methyltransferase (DNMT‑1) in MB (D324) cells, human umbilical vein endothelial cells (HUVECs) as well as in MB tissue sections. We hypothesized that infection and DNMT‑1 intracellular localization are linked. Uninfected and HCMV‑infected D324 cells and HUVECs were analyzed for HCMV immediate early (HCMV‑IE) protein, HCMV‑glycoproteinB (HCMV‑gB) and DNMT‑1 using immunofluorescence staining and quantitative ELISA. DNMT‑1 localized to the nucleus of uninfected and HCMV‑IE- expressing D324 cells and HUVECs, but accumulated in the extra nuclear space in all HCMV‑gB-positive cells. Inhibition of HCMV late protein expression by Cymevene® (ganciclovir) prevented the cytoplasmic localization of DNMT‑1. Treatment of HCMV‑ infected D324 cells and HUVECs with the methylation inhibitor 5-Azacytidine (5AZA), significantly increased HCMV‑IE and HCMV‑gB gene transcription and protein expression. Immunohistochemical staining of DNMT‑1 and HCMV proteins in MB cancer tissue sections revealed both nuclear and cytoplasmic DNMT‑1 localization. In conclusion, DNMT‑1 resides in the cytoplasm of HCMV‑gB-expressing HUVECs and D324 cells. Increased viral protein synthesis in 5AZA-treated cells suggests that HCMV replication may benefit from a DNA methyltransferase-free cellular environment. Our findings emphasize the importance of assessing potential viral activation in the treatment of MB patients with epigenetic drugs.

An Aptamer-Based Probe for Molecular Subtyping of Breast Cancer.

Molecular subtyping of breast cancer is of considerable interest owing to its potential for personalized therapy and prognosis. However, current methodologies cannot be used for precise subtyping, thereby posing a challenge in clinical practice. The aim of the present study is to develop a cell-specific single-stranded DNA (ssDNA) aptamer-based fluorescence probe for molecular subtyping of breast cancer.Methods: Cell-SELEX method was utilized to select DNA aptamers. Flow cytometry and confocal microscopy were used to study the specificity, binding affinity, temperature effect on the binding ability and target type analysis of the aptamers. In vitro and in vivo fluorescence imaging were used to distinguish the molecular subtypes of breast cancer cells, tissue sections and tumor-bearing mice.Results: Six SK-BR-3 breast cancer cell-specific ssDNA aptamers were evolved after successive in vitro selection over 21 rounds by Cell-SELEX. The Kd values of the selected aptamers were all in the low-nanomolar range, among which aptamer sk6 showed the lowest Kd of 0.61 ± 0.14 nM. Then, a truncated aptamer-based probe, sk6Ea, with only 53 nt and high specificity and binding affinity to the target cells was obtained. This aptamer-based probe was able to 1) differentiate SK-BR-3, MDA-MB-231, and MCF-7 breast cancer cells, as well as distinguish breast cancer cells from MCF-10A normal human mammary epithelial cells; 2) distinguish HER2-enriched breast cancer tissues from Luminal A, Luminal B, triple-negative breast cancer tissues, and adjacent normal breast tissues (ANBTs) in vitro; and 3) distinguish xenografts of SK-BR-3 tumor-bearing mice from those of MDA-MB-231 and MCF-7 tumor-bearing mice within 30 min in vivo.Conclusion: The results suggest that the aptamer-based probe is a powerful tool for fast and highly sensitive subtyping of breast cancer both in vitro and in vivo and is also very promising for the identification, diagnosis, and targeted therapy of breast cancer molecular subtypes.

Quantitative mass spectrometry imaging of glutathione in healthy and cancerous hen ovarian tissue sections by infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI).

A quantitative mass spectrometry imaging (QMSI) method for absolute quantification of glutathione (GSH) in healthy and cancerous hen ovarian tissues using infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) is presented. Using this technique, the ion abundance of GSH was normalized to that of a structural analogue, which was sprayed on the slide prior to mounting the tissue sections. This normalization strategy significantly improved the voxel-to-voxel variability; the variability is attributed to the overall ionization process. Subsequently, a series of calibration spots of stable isotope-labeled (SIL) GSH were pipetted on top of the tissue to construct a spatial calibration curve, and calculate the concentration of GSH in both tissue sections. The QMSI results were verified by LC-MS/MS quantification of GSH for the same tissues. GSH was extracted from tissue sections in a slightly acidic buffer and was then alkylated using N-ethylmaleimide to minimize autoxidation of GSH to glutathione disulfide. The alkylated GSH was separated from other contaminants using reversed phase liquid chromatography (RPLC) coupled to a triple quadrupole mass spectrometer, and the z-ion transition of NEM-GSH was used to quantify GSH in each tissue section. While the absolute values obtained using IR-MALDESI QMSI and LC-MS/MS were different, a ∼2-fold increase in the concentration of GSH in cancer tissue compared to the healthy tissue was observed using both techniques. Possible reasons for the difference between absolute concentration values obtained using IR-MALDESI QMSI and LC-MS/MS are also discussed.

Improved detectability of sex steroids from frozen sections of breast cancer tissue using GC-triple quadrupole-MS

Sex steroids in clinical endocrinology have been mainly investigated with peripheral blood and urine samples, while there is limited information regarding the local levels within tissues. To improve analytical properties of sex steroids from trace amounts of tissue samples, two-phase extractive ethoxycarbonlyation and subsequent pentafluoropropionyl derivatization coupled to gas chromatography–tandem mass spectrometry (GC–MS/MS) was developed. The optimized analytical conditions led to excellent chromatographic separation of 15 estrogens, 6 androgens, and 2 progestins. The quantitative results were calculated based on in-house control samples as the steroid-free tissues, and the precision and accuracy were 4.2%–26.8% and 90.8%–116.4%, respectively. The on-column limit of quantification was from 180 fg to 0.5 pg for androgens and estrogens, and 1.25 pg for progestins, which were found to be linear (r2 > 0.990). The validated method was then applied to quantify 7 sex steroids from three 100-μm-thick frozen breast tissue slices from postmenopausal patients with breast cancer. This is the first report on the improved GC–MS/MS method for the detection of androgens and pregnenolone from breast cancer tissues, and it can be a useful technique to measure the local levels of sex steroids, thus, enhancing our understanding of the pathophysiological significances of steroidogenesis.

A novel histological examination with dynamic three-dimensional reconstruction from multiple immunohistochemically stained sections of a PD-L1-positive colon cancer.

Programmed cell death-ligand 1 (PD-L1) expression is observed in patients with microsatellite instability-high (MSI-H) colon cancer, which is susceptible to immune checkpoint blockade. The aim of this study was to investigate the interrelationship between PD-L1-positive cells and cytotoxic T cells, lymphatic vessels and vascular endothelium by using histological examination with the three-dimensional (3D) reconstruction of a PD-L1-positive colon cancer. Serial sections of MSI-H colon cancer tissue were stained with haematoxylin and eosin (H&E) and Masson trichrome stains; immunohistochemical analysis of PD-L1, CD8, D2-40 and CD31 was performed. Several 3D models of MSI-H colon cancer were reconstructed with a 3D data visualisation system. Moreover, 18 serial sections were stained with PD-L1, cytokeratin AE1/AE3, CD45, CD31, CD68 and H&E in the same case to confirm that PD-L1 was expressed on tumour cells, CD31-positive cells and macrophages in the invasive frontal region. Notably, there was a peak in the expression of PD-L1 and CD31 in the invasive frontal region. D2-40-positive cells were abundant in the overall tumour stroma, and CD8-positive cells infiltrated the tumour parenchyma. PD-L1 was expressed on tumour cells in the parenchyma and other cells in the stroma. Additional staining of 18 consecutive sections revealed that the other cells were CD68-positive and CD45-positive macrophages and CD31-positive proliferating vascular endothelial cells. We confirmed that PD-L1 was highly expressed in the invasive frontal region in 3D models of MSI-H colon cancer tissue. This method can be useful for accurately evaluating the localisation of immune checkpoint molecules.

Isolation and characterization of a new naturally immortalized human breast carcinoma cell line, KAIMRC1

BackgroundBreast cancer is one of the most common cancer and a leading cause of death in women. Up to date the most commonly used breast cancer cell lines are originating from Caucasians or Afro-Americans but rarely cells are being derived from other ethnic groups. Here we describe for the first time the establishment of a naturally transformed breast cancer cell line, KAIMRC1 from an Arab woman of age 62 suffering from stage IIB breast cancer (T2N1M0). Moreover, we have characterized these cells for the biological and molecular markers, induction of MAPK pathways as well as its response to different commercially available drugs and compounds.MethodsBreast cancer tissue sections were minced and cultured in media for several weeks. KAIMRC1 cells were successfully isolated from one of the primary breast tumor tissue cultures without any enzymatic digestion. To study the growth characteristics of the cells, wound healing assay, clonogenic assay, cell proliferation assays and live cell time-lapse microscopy was performed. Karyotyping, Immunophenotyping and molecular pathway specific compound treatment was also performed. A selective breast cancer gene expression panel was used to identify genes involved in the signal transduction dysregulation and malfunction of normal biological processes during breast carcinogenesis.ResultsThese cells are ER/PR-positive and HER2-negative. The epithelial nature of these cells was confirmed by flow cytometry analysis using epithelial cell markers. They are cuboidal in shape and relatively smaller in size as compared to established cell lines, MCF-7, MDA MB-231 and the normal breast cell line, MCF-10A. In normal cell culture conditions these cells showed the capability of growing both in monolayer as well as in 3-D conformation. They showed a doubling time in vitro of approximately 24 h. They exhibit a modal karyotype of 58–63,X with abnormalities in a couple of chromosomes. KAIMRC1 cells were found to be more responsive to drug treatment in vitro in comparison to the established MDA MB-231 and MCF-7 cell lines.ConclusionsIn conclusion we have isolated and characterized a new naturally immortalized breast cell line, KAIMRC1 with a potential to play a key role in opening up novel avenues towards the understanding of breast carcinoma.

Analyte-localization device for point-of-use processing of sub-millimetre areas on surfaces

We present a portable, simple-to-operate, point-of-use analyte surface localization device. We take advantage of a set of hydrodynamic design features and components that achieve passive analyte localization by means of a single vacuum input. The vacuum source can be supplied by mechanical or battery-operated vacuum sources that are portable and allow point-of-use operation in the absence of electricity. We discuss the governing hydrodynamic principle and design parameters in detail. In a case study, we demonstrate the applicability of our technology to successfully localize a solution of rhodamine on a polydimethylsiloxane (PDMS) substrate and produce sub-millimetre-sized spots via application of a mild vacuum pressure of less than 10 kPa. In addition, we demonstrate local staining of breast cancer cell blocks and on human breast cancer tissue sections.

Regulator of G-protein signaling 5 enhances portal vein invasion in hepatocellular carcinoma.

Portal vein invasion (PVI) is a major prognostic factor in hepatocellular carcinoma (HCC). The aim of the present study was to identify molecules that regulate PVI. Sections of cancerous tissue, paired noncancerous tissue and the PVI area were collected from 3 frozen HCC sections, using laser microdissection. The present study focused on 3 upregulated molecules, integrin β3 (ITGB3), secreted phosphoprotein 1 (SPP1) and regulator of G-protein signaling 5 (RGS5), and 2 molecules that were downregulated in PVI tissue compared with cancer tissue, metallothionein 1G (MT1G) and metallothionein 1H (MT1H), as determined by cDNA microarray analysis. Reverse transcription-quantitative polymerase chain reaction analysis of 32 HCC cases revealed that RGS5 mRNA levels were significantly increased and MT1 G and MT1H mRNA levels were significantly decreased in cancerous tissue compared with noncancerous tissue. However, there was no significant difference in ITGB3 and SPP1 expression. There were no significant differences between the expression of these molecules and any clinicopathologic factors, including PVI. Immunohistochemical staining for RGS5 in 60 HCC cases demonstrated that RGS5 protein levels were higher in cancerous tissue compared with paired noncancerous tissue in 63.3% of HCC cases. Furthermore, high expression of RGS5 in cancerous tissue was significantly associated with PVI and tended to be associated with intrahepatic metastasis. Confluent multinodular type was significantly more frequent in cases with high expression of RGS5 in the cancerous tissue. Therefore, RGS5 may be a useful prognostic biomarker as well as a potential target of molecular therapy to treat HCC.

Phospholipase D inhibitors reduce human prostate cancer cell proliferation and colony formation

Background:Phospholipases D1 and D2 (PLD1/2) hydrolyse cell membrane glycerophospholipids to generate phosphatidic acid, a signalling lipid, which regulates cell growth and cancer progression through effects on mTOR and PKB/Akt. PLD expression and/or activity is raised in breast, colorectal, gastric, kidney and thyroid carcinomas but its role in prostate cancer (PCa), the major cancer of men in the western world, is unclear.Methods:PLD1 protein expression in cultured PNT2C2, PNT1A, P4E6, LNCaP, PC3, PC3M, VCaP, 22RV1 cell lines and patient-derived PCa cells was analysed by western blotting. PLD1 protein localisation in normal, benign prostatic hyperplasia (BPH), and castrate-resistant prostate cancer (CRPC) tissue sections and in a PCa tissue microarray (TMA) was examined by immunohistochemistry. PLD activity in PCa tissue was assayed using an Amplex Red method. The effect of PLD inhibitors on PCa cell viability was measured using MTS and colony forming assays.Results:PLD1 protein expression was low in the luminal prostate cell lines (LNCaP, VCaP, 22RV1) compared with basal lines (PC3 and PC3M). PLD1 protein expression was elevated in BPH biopsy tissue relative to normal and PCa samples. In normal and BPH tissue, PLD1 was predominantly detected in basal cells as well in some stromal cells, rather than in luminal cells. In PCa tissue, luminal cells expressed PLD1. In a PCa TMA, the mean peroxidase intensity per DAB-stained Gleason 6 and 7 tissue section was significantly higher than in sections graded Gleason 9. In CRPC tissue, PLD1 was expressed prominently in the stromal compartment, in luminal cells in occasional glands and in an expanding population of cells that co-expressed chromogranin A and neurone-specific enolase. Levels of PLD activity in normal and PCa tissue samples were similar. A specific PLD1 inhibitor markedly reduced the survival of both prostate cell lines and patient-derived PCa cells compared with two dual PLD1/PLD2 inhibitors. Short-term exposure of PCa cells to the same specific PLD1 inhibitor significantly reduced colony formation.Conclusions:A new specific inhibitor of PLD1, which is well tolerated in mice, reduces PCa cell survival and thus has potential as a novel therapeutic agent to reduce prostate cancer progression. Increased PLD1 expression may contribute to the hyperplasia characteristic of BPH and in the progression of castrate-resistant PCa, where an expanding population of neuroendocrine-like cells express PLD1.

Immunohistochemical evalulation of activated Ras and Rac1 as potential downstream effectors of aquaporin-5 in breast cancer in vivo

Aberrant levels of aquaporin-5 (AQP5) expression have been observed in several types of cancer, including breast cancer, where AQP5 overexpression is associated with metastasis and poor prognosis. In cultured cancer cells, AQP5 facilitates cell migration and activates Ras signaling. Both increased cell migration and Ras activation are associated with cancer metastasis, but so far it is unknown if AQP5 also affects these processes in vivo. Therefore, we investigated if high AQP5 expression in breast cancer tissue correlated with increased activation of Ras and of Rac1, which is a GTPase also involved in cell migration. This was accomplished by immunohistochemical analysis of invasive ductal carcinoma of breast tissue sections from human patients, followed by qualitative and quantitative correlation analysis between AQP5 and activated Ras and Rac1. Immunohistochemistry revealed that activation of Ras and Rac1 was positively correlated. There was, however, no correlation between high AQP5 expression and activation of Ras, whereas a nonsignificant, but positive, tendency between the levels of AQP5 and activated Rac1 levels was observed. In summary, this is the first report that correlates AQP5 expression levels to downstream signaling partners in breast cancer tissue sections. The results suggest Rac1 as a potential downstream signaling partner of AQP5 in vivo.

Expression of circadian clock genes in human colorectal adenoma and carcinoma.

Circadian rhythms are fundamental biological systems in most organisms. Epidemiological and animal studies have demonstrated that disruption of circadian rhythms is linked to tumor progression and mammalian tumorigenesis. However, the clinical significance of in situ clock gene expression in precancerous and cancerous colorectal lesions remains unknown. The present study aimed to investigate mRNA transcript levels of circadian clock genes within human colorectal cancer and adenoma tissue sections. Using in situ hybridization, the expression of key clock genes, including period circadian protein homolog (Per) 1 and 2, cryptochrome 1 (Cry1), circadian locomoter output cycles protein kaput (Clock), brain and muscle ARNT-like protein 1 (Bmal1) and casein kinase 1ε (CK1ε) were retrospectively examined in 51 cases of colorectal carcinoma and 10 cases of adenoma. The expression of clock genes was almost undetectable in the majority of adenomas, whereas positive expression of clock genes was observed in 27–47% of carcinomas. Notably, positive Per1, Per2 and Clock staining in colorectal carcinomas were each significantly associated with a larger tumor size (P=0.012, P=0.011 and P=0.009, respectively). Tumors with positive Per2 and Clock expression tended to exhibit deeper depth of invasion and were generally more advanced than tumors that did not express these genes (P=0.052 and P=0.064, respectively). However, no statistically significant association was observed between clock gene expression and clinicopathological variables, including histopathological differentiation, lymph node metastasis, depth of invasion or disease stage, although Per2-positive tumors tended to be associated with poorer overall survival (P=0.060). The results of the current study suggest that dysregulated expression of clock genes may be important in human colorectal tumorigenesis.