Systems pathology by multiplexed immunohistochemistry and whole-slide digital image analysis

The paradigm of molecular histopathology is shifting from a single-marker immunohistochemistry towards multiplexed detection of markers to better understand the complex pathological processes. However, there are no systems allowing multiplexed IHC (mIHC) with high-resolution whole-slide tissue imaging and analysis, yet providing feasible throughput for routine use. We present an mIHC platform combining fluorescent and chromogenic staining with automated whole-slide imaging and integrated whole-slide image analysis, enabling simultaneous detection of six protein markers and nuclei, and automatic quantification and classification of hundreds of thousands of cells in situ in formalin-fixed paraffin-embedded tissues. In the first proof-of-concept, we detected immune cells at cell-level resolution (n = 128,894 cells) in human prostate cancer, and analysed T cell subpopulations in different tumour compartments (epithelium vs. stroma). In the second proof-of-concept, we demonstrated an automatic classification of epithelial cell populations (n = 83,558) and glands (benign vs. cancer) in prostate cancer with simultaneous analysis of androgen receptor (AR) and alpha-methylacyl-CoA (AMACR) expression at cell-level resolution. We conclude that the open-source combination of 8-plex mIHC detection, whole-slide image acquisition and analysis provides a robust tool allowing quantitative, spatially resolved whole-slide tissue cytometry directly in formalin-fixed human tumour tissues for improved characterization of histology and the tumour microenvironment.Peer reviewe

An Activatable Cancer-Targeted Hydrogen Peroxide Probe for Photoacoustic and Fluorescence Imaging.

Reactive oxygen species play an important role in cancer, however, their promiscuous reactivity, low abundance, and short-lived nature limit our ability to study them in real time in living subjects with conventional noninvasive imaging methods. Photoacoustic imaging is an emerging modality for in vivo visualization of molecular processes with deep tissue penetration and high spatiotemporal resolution. Here, we describe the design and synthesis of a targeted, activatable probe for photoacoustic imaging, which is responsive to one of the major and abundant reactive oxygen species, hydrogen peroxide (H2O2). This bifunctional probe, which is also detectable with fluorescence imaging, is composed of a heptamethine carbocyanine dye scaffold for signal generation, a 2-deoxyglucose cancer localization moiety, and a boronic ester functionality that specifically detects and reacts to H2O2. The optical properties of the probe were characterized using absorption, fluorescence, and photoacoustic measurements; upon addition of pathophysiologic H2O2 concentrations, a clear increase in fluorescence and red-shift of the absorption and photoacoustic spectra were observed. Studies performed in vitro showed no significant toxicity and specific uptake of the probe into the cytosol in breast cancer cell lines. Importantly, intravenous injection of the probe led to targeted uptake and accumulation in solid tumors, which enabled noninvasive photoacoustic and fluorescence imaging of H2O2. In conclusion, the reported probe shows promise for the in vivo visualization of hydrogen peroxide. SIGNIFICANCE: This study presents the first activatable and cancer-targeted hydrogen peroxide probe for photoacoustic molecular imaging, paving the way for visualization of hydrogen peroxide at high spatiotemporal resolution in living subjects.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/20/5407/F1.large.jpg

Quantitative Analysis of Immune Infiltrates in Primary Melanoma.

Novel methods to analyze the tumor microenvironment (TME) are urgently needed to stratify melanoma patients for adjuvant immunotherapy. Tumor-infiltrating lymphocyte (TIL) analysis, by conventional pathologic methods, is predictive but is insufficiently precise for clinical application. Quantitative multiplex immunofluorescence (qmIF) allows for evaluation of the TME using multiparameter phenotyping, tissue segmentation, and quantitative spatial analysis (qSA). Given that CD3+CD8+ cytotoxic lymphocytes (CTLs) promote antitumor immunity, whereas CD68+ macrophages impair immunity, we hypothesized that quantification and spatial analysis of macrophages and CTLs would correlate with clinical outcome. We applied qmIF to 104 primary stage II to III melanoma tumors and found that CTLs were closer in proximity to activated (CD68+HLA-DR+) macrophages than nonactivated (CD68+HLA-DR-) macrophages (P < 0.0001). CTLs were further in proximity from proliferating SOX10+ melanoma cells than nonproliferating ones (P < 0.0001). In 64 patients with known cause of death, we found that high CTL and low macrophage density in the stroma (P = 0.0038 and P = 0.0006, respectively) correlated with disease-specific survival (DSS), but the correlation was less significant for CTL and macrophage density in the tumor (P = 0.0147 and P = 0.0426, respectively). DSS correlation was strongest for stromal HLA-DR+ CTLs (P = 0.0005). CTL distance to HLA-DR- macrophages associated with poor DSS (P = 0.0016), whereas distance to Ki67- tumor cells associated inversely with DSS (P = 0.0006). A low CTL/macrophage ratio in the stroma conferred a hazard ratio (HR) of 3.719 for death from melanoma and correlated with shortened overall survival (OS) in the complete 104 patient cohort by Cox analysis (P = 0.009) and merits further development as a biomarker for clinical application

Multiplex immunohistochemical analysis of granulomatous inflammation in lung tissue sections using a mouse model of M. avium infection

INTRODUCTION: Investigating mechanisms of how intracellular bacterial pathogens such as Mycobacterium. avium (M. avium) evade the host immune response and replicate within macrophages is crucial to devising rational targets for host-directed therapies (HDT) against these associated diseases. This studied utilized the congenic mouse strain B6.Sst1S, which contains the super-susceptibility to tuberculosis (TB) allele. Among murine models of TB, this strain uniquely replicates human disease because mice develop granulomas with central caseous necrosis. Utilizing a susceptible model for M. avium infection, this study investigated the effect of mycobacterial pathogenesis on altering macrophage phenotypes and T cells distribution in areas of pulmonary granulomatous inflammation. METHODS:12 formalin fixed paraffin embedded (FFPE) lung sections from M. avium infected B6.Sst1S and B6 mice were examined microscopically (12 weeks post infection (wpi) n=5, 16 wpi=7). A targeted histology approach was initiated by using MRI coordinates to dictate the depths at which formalin fixed paraffin embedded (FFPE) lung samples were sectioned. Since interpretation of MRI images displayed no evidence of 2 discrete necrotizing granulomas, lungs were cut at sections representative of diffuse pathology at 2 mm into FFPE blocks. Using the Opal MethodTM (Akoya Biosciences), 6- plex immunohistochemical staining was performed with Arginase-1 (Arg1), inducible nitric oxide synthase (iNOS), CD68, CD3, M. tuberculosis antigen (cross-reacts with M. avium) and DAPI to segment nuclei. Slides were digitized by a Vectra PolarisTM fluorescent whole slide scanner. Autofluorescence was removed by InFormTM, and image analysis (IA) was conducted using HaloTM IA software. Statistical analysis was conducted using GraphPad PrismTM 8.0. RESULTS: Sst1 mediated susceptibility was statistically evident at 16 wpi but not at 12 wpi. B6.Sst1S mice showed a statistically significant (P <0.05) increase in M. avium+ cell expression in the non-inoculated lung lobes, but not the inoculated lung lobes. Pulmonary lesions within the inoculated and non-inoculated lung lobes contain different immune signatures. The predominately primary lesions of the inoculated lung lobes were associated with increased CD3+, M. avium+, and iNOS+ cell levels. When controlling for level of infection, there was lower levels of CD3+ cells within granulomatous lesions of B6.Sst1S mice, especially in the non-inoculated lung lobe. Controlling for level of infection also revealed elevated iNOS+ M. avium- cell expression in B6 mice. We observed elevated Arg1+ cell expression near iNOS+ M. avium+ cells, and, qualitatively, around larger lesions. T cell proximity analysis was contradictory and offers lessons for future the development of future IA modules. CONCLUSIONS: Sst1 mediated susceptibility was evident at 16 wpi and predominately mediated through secondary, metastatic lesions. Sst1 mediated susceptibility was also associated with fewer supportive cells (T cells and iNOS+ M. avium- cells) within granulomatous lesions. Future studies are necessary to evaluate to what degree granulomatous lesion Arg1+ cell expression and CD3+ proximity correlate to susceptibility

Nanoparticle-Enabled In Vivo Photoacoustic Molecular Imaging of Cancer

Photoacoustic (PA) imaging is an emerging biomedical imaging modality that combines optical and ultrasound imaging technologies. PA imaging relies on the absorption of electromagnetic energy (usually in the form of visible or near-infrared light) leading to the generation of acoustic waves by thermoelastic expansion, which can be detected with an ultrasound detector. PA imaging can be used to detect endogenous chromophores such as deoxyhemoglobin and oxyhemoglobin, or can be used together with external nanosensors for added functionality. The former is used to measure things like blood oxygenation, while the latter opens up many possibilities for PA imaging, limited only to the availability of optical nanosensors. In this dissertation, I employ the use of PA nanosensors for contrast enhancement and molecular imaging in in vivo small animal cancer models. In the first section, I introduce a novel PA background reduction technique called the transient triplet differential (TTD) method. The TTD method exploits the fact that phosphorescent dyes possess a triplet state with a unique red-shifted absorption wavelength, distinct from its ordinary singlet state absorption profile. By pumping these dyes into the triplet state and comparing the signal to the unpumped dyes, a differential signal can be obtained which solely originates from these dyes. Since intrinsic chromophores of biological tissue are not able to undergo intersystem crossing and enter the triplet state, the TTD method can facilitate “true” background free molecular imaging by excluding the signals from every other chromophore outside the phosphorescent dye. Here, I demonstrate up to an order of magnitude better sensitivity of the TTD method compared to other existing contrast enhancement techniques in both in vitro experiments and in vivo cancer models. In the second section, I explore the use of a nanoparticle formulation of a repurposed FDA-approved drug called clofazimine for diagnosis of prostate cancer. Clofazimine nanoparticles have a high optical absorbance at 495 nm and has been known to specifically accumulate in macrophages as they form stable crystal-like inclusions once they are uptaken by macrophages. Due to the presence of tumor associated macrophages, it is expected that clofazimine would accumulate in much higher quantities in the cancerous prostate compared to normal prostates. Here, I show that there was indeed a significantly higher accumulation of clofazimine nanoparticles in cancerous prostates compared to normal prostates in a transgenic mouse model, which was detectable both using histology and ex vivo PA imaging. In the third and final section, I explore the use of a potassium (K+) nanosensor together with PA imaging in measuring the in vivo K+ distribution in the tumor microenvironment (TME). K+ is the most abundant ion in the body and has recently been shown to be at a significantly higher concentration in the tumor. The reported 5-10 fold elevation (25-50 mM compared to 5mM) in the tumor has been shown to inhibit immune cell efficacy, and thus immunotherapy. Despite the abundance and importance of K+ in the body, few ways exist to measure it in vivo. In this study, a solvatochromic dye K+ nanoparticle (SDKNP) was used together with PA imaging to quantitatively measure the in vivo distribution of K+ in the TME. Significantly elevated K+ levels were found in the TME, with an average concentration of approximately 29 mM, matching the values found by the previous study. The results were then verified using mass spectrometry.PHDBiomedical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/155291/1/tanjoel_1.pd

Uncovering cancer metabolic signatures by high-content stimulated Raman scattering (SRS) imaging

Cancer is still one of the most serious health problems worldwide and cancer resistance to chemotherapy, the most wildly use therapeutic strategy for cancer, mounts the biggest challenges for current anti-cancer treatment. The unique characteristics of chemo-resistant cancer cell such as metabolic hallmark can largely facilitate surmounting this difficulty by serving as a therapeutic target to fight against chemo-resistance. However, the understanding of cancer metabolism is still limited, partly resulting from the lack of suitable analytic approaches. My dissertation work applied recently developed stimulated Raman scattering (SRS) imaging on cancer cells to uncover their metabolic signatures for the development of more effective cancer therapy. Taking advantage of SRS imaging, we uncovered that cisplatin-resistant cell have increased fatty acid (FA) uptake, accompanied with reduced glucose uptake and lipogenesis. This metabolic reprograming from glucose to FA dependent anabolic and energy metabolism enables us to develop a rapid diagnostic method for cisplatin-resistance and a therapeutic strategy for cisplatin-resistant cancer. Moreover, we used SRS imaging to estimate the ratio of saturated (SFAs) and unsaturated fatty acids (UFAs) in cancer cell and revealed the role of Stearoyl Co-A desaturase 1 (SCD) on maintaining the intracellular balance of SFAs and UFAs. The unbalance SFAs/UFAs leaded to endoplasmic reticulum (ER) stress, presented as stiff and disorganized ER structure in SRS imaging. This ER stress induced cancer cell apoptosis in vitro, suggesting the therapeutic potential of targeting the lipid balance. To further dissect the metabolic features and reprograming in cancer cells, we developed a high-content hyperspectral SRS (h2SRS) imaging approach by introducing sparsity-driven hyperspectral image decomposition to SRS image post-processing. h2SRS can simultaneously map five major biomolecules involving protein, carbohydrate, FA, cholesterol, and nucleic acid at the single cell level, revealing the acute and adapted metabolic reprograming induced by chemotherapy in cancer cells. This approach accelerates the discoveries of new therapeutic targets against chemo-resistance and benefit the exploration of cellular metabolism study

Multiplexed High-Resolution Imaging Approach to Decipher the Cellular Heterogeneity of the Kidney and its Alteration in Kidney Disease and Nephrolithiasis

Indiana University-Purdue University Indianapolis (IUPUI)Kidney disease and nephrolithiasis both present a major burden on the health care system in the US and worldwide. The cellular and molecular events governing the pathogenesis of these diseases are not fully understood. We propose that defining the cellular heterogeneity and niches in human and mouse kidney tissue specimens from controls and various models of renal disease could provide unique insights into the molecular pathogenesis. For that purpose, a multiplexed fluorescence imaging approach using co-detection by Indexing (CODEX) was used, using a panel of 33 and 38 markers for mouse and human kidney tissues, respectively. A customized computational analytical pipeline was developed and applied to the imaging data using unsupervised and/or semi-supervised machine learning and statistical approaches. The goal was to identify various cell populations present within the tissues, as well as identify unique cellular niches that may be altered with disease and/or injury. In mice, we examined disease models of acute kidney injury (AKI) and in human tissues we analyzed specimens from patients with AKI, IgA nephropathy, chronic kidney disease, systemic lupus erythematosus, and nephrolithiasis. In both mice and humans, the disease and reference samples show similar broad cell populations for the main segments of the nephron, endothelium, as well as similar groups of immune cells, such as resident macrophages and neutrophils. When comparing between health and disease, however, a change in the distribution of few sub-populations occurred. For example, in human kidney tissues, the abundance and distribution of a subpopulation of proximal tubules positive for THY1 (a marker of differentiation and repair), was markedly reduced with disease. Changes observed in mouse tissues included shifts in the immune cell population types and niches with disease. We propose that our analytical workflow and the observed changes in situ will play an important role in deciphering the pathogenesis of kidney disease