Spatial mapping of the collagen distribution in human and mouse tissues by force volume atomic force microscopy

Changes in the elastic properties of living tissues during normal development and in pathological processes are often due to modifications of the collagen component of the extracellular matrix at various length scales. Force volume AFM can precisely capture the mechanical properties of biological samples with force sensitivity and spatial resolution. The integration of AFM data with data of the molecular composition contributes to understanding the interplay between tissue biochemistry, organization and function. The detection of micrometer-size, heterogeneous domains at different elastic moduli in tissue sections by AFM has remained elusive so far, due to the lack of correlations with histological, optical and biochemical assessments. In this work, force volume AFM is used to identify collagen-enriched domains, naturally present in human and mouse tissues, by their elastic modulus. Collagen identification is obtained in a robust way and affordable timescales, through an optimal design of the sample preparation method and AFM parameters for faster scan with micrometer resolution. The choice of a separate reference sample stained for collagen allows correlating elastic modulus with collagen amount and position with high statistical significance. The proposed preparation method ensures safe handling of the tissue sections guarantees the preservation of their micromechanical characteristics over time and makes it much easier to perform correlation experiments with different biomarkers independently

Deconvoluting hepatic processing of carbon nanotubes

Single-wall carbon nanotubes present unique opportunities for drug delivery, but have not advanced into the clinic. Differential nanotube accretion and clearance from critical organs have been observed, but the mechanism not fully elucidated. The liver has a complex cellular composition that regulates a range of metabolic functions and coincidently accumulates most particulate drugs. Here we provide the unexpected details of hepatic processing of covalently functionalized nanotubes including receptor-mediated endocytosis, cellular trafficking and biliary elimination. Ammonium-functionalized fibrillar nanocarbon is found to preferentially localize in the fenestrated sinusoidal endothelium of the liver but not resident macrophages. Stabilin receptors mediate the endocytic clearance of nanotubes. Biocompatibility is evidenced by the absence of cell death and no immune cell infiltration. Towards clinical application of this platform, nanotubes were evaluated for the first time in non-human primates. The pharmacologic profile in cynomolgus monkeys is equivalent to what was reported in mice and suggests that nanotubes should behave similarly in humans

Tunneling Nanotubes Provide a Unique Conduit for Intercellular Transfer of Cellular Contents in Human Malignant Pleural Mesothelioma

Tunneling nanotubes are long, non-adherent F-actin-based cytoplasmic extensions which connect proximal or distant cells and facilitate intercellular transfer. The identification of nanotubes has been limited to cell lines, and their role in cancer remains unclear. We detected tunneling nanotubes in mesothelioma cell lines and primary human mesothelioma cells. Using a low serum, hyperglycemic, acidic growth medium, we stimulated nanotube formation and bidirectional transfer of vesicles, proteins, and mitochondria between cells. Notably, nanotubes developed between malignant cells or between normal mesothelial cells, but not between malignant and normal cells. Immunofluorescent staining revealed their actin-based assembly and structure. Metformin and an mTor inhibitor, Everolimus, effectively suppressed nanotube formation. Confocal microscopy with 3-dimensional reconstructions of sectioned surgical specimens demonstrated for the first time the presence of nanotubes in human mesothelioma and lung adenocarcinoma tumor specimens. We provide the first evidence of tunneling nanotubes in human primary tumors and cancer cells and propose that these structures play an important role in cancer cell pathogenesis and invasion

Understanding the three-dimensional world from two-dimensional immunofluorescent adjacent sections

Visualizing tissue structures in three-dimensions (3D) is crucial to understanding normal and pathological phenomena. However, staining and imaging of thick sections and whole mount samples can be challenging. For decades, researchers have serially sectioned large tissues and painstakingly reconstructed the 3D volume. Advances in automation, from sectioning to alignment, now greatly accelerate the process. In addition, immunofluorescent staining methods allow multiple antigens to be simultaneously detected and analyzed volumetrically. The objective was to incorporate multi-channel immunofluorescent staining and automation in 3D reconstruction of serial sections for volumetric analysis. Paraffin-embedded samples were sectioned manually but were processed, stained, imaged and aligned in an automated fashion. Reconstructed stacks were quantitatively analyzed in 3D. By combining automated immunofluorescent staining and tried-and-true methods of reconstructing adjacent sections, we were able to visualize, in detail, not only the geometric structures of the sample but also the presence and interactions of multiple proteins and molecules of interest within their 3D environment. Advances in technology and software algorithms have significantly expedited the 3D reconstruction of serial sections. Automated, multi-antigen immunofluorescent staining will significantly broaden the range and complexity of scientific questions that can be answered with this methodology

PI3K pathway regulates survival of cancer stem cells residing in the perivascular niche following radiation in medulloblastoma in vivo

Medulloblastomas are brain tumors that arise in the cerebellum of children and contain stem cells in a perivascular niche thought to give rise to recurrence following radiation. We used several mouse models of medulloblastomas in parallel to better understand how the critical cell types in these tumors respond to therapy. In our models, the proliferating cells in the tumor bulk undergo radiation-induced, p53-dependent apoptotic cell death. Activation of Akt signaling via PTEN loss transforms these cells to a nonproliferating extensive nodularity morphology. By contrast, the nestin-expressing perivascular stem cells survive radiation, activate PI3K/Akt pathway, undergo p53-dependent cell cycle arrest, and re-enter the cell cycle at 72 h. Furthermore, the ability of these cells to induce p53 is dependent on the presence of PTEN. These cellular characteristics are similar to human medulloblastomas. Finally, inhibition of Akt signaling sensitizes cells in the perivascular region to radiation-induced apoptosis

Anti gC1qR/p32/HABP1 Antibody Therapy Decreases Tumor Growth in an Orthotopic Murine Xenotransplant Model of Triple Negative Breast Cancer

gC1qR is highly expressed in breast cancer and plays a role in cancer cell proliferation. This study explored therapy with gC1qR monoclonal antibody 60.11, directed against the C1q binding domain of gC1qR, in a murine orthotopic xenotransplant model of triple negative breast cancer. MDA231 breast cancer cells were injected into the mammary fat pad of athymic nu/nu female mice. Mice were segregated into three groups (n = 5, each) and treated with the vehicle (group 1) or gC1qR antibody 60.11 (100 mg/kg) twice weekly, starting at day 3 post-implantation (group 2) or when the tumor volume reached 100 mm3 (group 3). At study termination (d = 35), the average tumor volume in the control group measured 895 ± 143 mm3, compared to 401 ± 48 mm3 and 701 ± 100 mm3 in groups 2 and 3, respectively (p < 0.05). Immunohistochemical staining of excised tumors revealed increased apoptosis (caspase 3 and TUNEL staining) in 60.11-treated mice compared to controls, and decreased angiogenesis (CD31 staining). Slightly decreased white blood cell counts were noted in 60.11-treated mice. Otherwise, no overt toxicities were observed. These data are the first to demonstrate an in vivo anti-tumor effect of 60.11 therapy in a mouse model of triple negative breast cancer

Three-Dimensional Histologic, Immunohistochemical and Multiplex Immunofluorescence Analysis of Dynamic Vessel Co-Option of Spread Through Air Spaces (STAS) in Lung Adenocarcinoma

BACKGROUND: Spread through air spaces (STAS) is a method of invasion in lung adenocarcinoma, associated with tumor recurrence and poor survival. The spatial orientation of STAS cells/clusters to the lung alveolar parenchyma is not known. The aim of this study was to utilize high resolution and high-quality three-dimensional (3D) reconstruction of images from immunohistochemistry (IHC) and multiplex immunofluorescence (IF) experiments to understand the spatial architecture of tumor cell clusters by STAS in the lung parenchyma. METHODS: Four lung adenocarcinomas: 3 micropapillary (MIP) predominant and 1 solid (SN) predominant adenocarcinoma subtypes, were investigated. A 3D reconstruction image was created from the formalin fixed paraffin-embedded (FFPE) blocks. 350 serial sections were obtained and stained with hematoxylin and eosin (H&E) (100 slides), IHC (200 slides), and multiplex IF (50 slides) with the following antibodies: CD31, collagen type 4, TTF-1 and E-Cadherin. Whole slide images (WSIs) were reconstructed into 3D images for evaluation. RESULTS: Serial 3D image analysis by H&E as well as IHC and IF showed the MIP clusters and SN nests of STAS focally attached to alveolar walls away from the main tumor. CONCLUSION: Our 3-D reconstructions demonstrated STAS tumor cells can attach to alveolar walls rather than appearing free floating as seen on 2D sections. This suggests that tumor cells detach from the main tumor, migrate through air spaces and reattach to alveolar walls through vessel co-option allowing them to survive and grow. This may explain the higher recurrence rate and worse survival for STAS positive tumors undergoing limited resection compared to lobectomy

Intercellular Transfer of Oncogenic KRAS via Tunneling Nanotubes Introduces Intracellular Mutational Heterogeneity in Colon Cancer Cells

Mutated forms of the RAS oncogene drive 30% of all cancers, but they cannot be targeted therapeutically using currently available drugs. The molecular and cellular mechanisms that create a heterogenous tumor environment harboring both mutant and wild-type RAS have not been elucidated. In this study, we examined horizontal transfer of mutant KRAS between colorectal cancer (CRC) cells via a direct form of cell-to-cell communication called tunneling nanotubes (TNTs). TNT formation was significantly higher in CRC cell lines expressing mutant KRAS than CRC cell lines expressing wild-type RAS; this effect was most pronounced in metastatic CRC cell lines with both mutant KRAS and deficiency in mismatch repair proteins. Using inverted and confocal fluorescence time-lapse and fluorescence recovery after photobleaching (FRAP)-based microscopy, we observed GFP-tagged mutant KRASG12D protein trafficking between CRC cells through TNTs within a span of seconds to several minutes. Notably, acquisition of mutant KRAS increased Extracellular Signal-regulated Kinase (ERK) phosphorylation and upregulated tunneling nanotube formation in recipient wildtype CRC cells. In conclusion, these findings suggest that intercellular horizontal transfer of RAS can occur by TNTs. We propose that intercellular transfer of mutant RAS can potentially induce intratumoral heterogeneity and result in a more invasive phenotype in recipient cells