Serial Tissue Sections Research Articles

Cross-sectional study to evaluate the utility of elastic tissue staining in primary cicatricial alopecia

Background and aimsDiagnosing end-stage primary cicatricial alopecia (PCA) on routine histology is challenging since the major diagnostic feature (inflammatory infiltrate) may be minimal or absent. This study aimed to assess...

Immune Cell Densities Predict Response to Immune Checkpoint-Blockade in Head and Neck Cancer.

Pre-ICB tumor tissue sections were obtained from 9 responders (complete response, partial response, or stable disease) and 11 non-responders (progressive disease) classified via RECISTv1.1. A custom multi-immunofluorescence (mIF) staining assay was designed, optimized, and applied to characterize tumor cells (pan-cytokeratin), T cells (CD4, CD8), B cells (CD19, CD20), myeloid cells (CD16, CD56, CD163), dendritic cells (LAMP3), fibroblasts (alpha-Smooth Muscle Actin), proliferative status (Ki67) and immunoregulatory molecules (PD1). Spatial metrics were compared among groups. Serial tissue sections were scored for TLS in both H&E and mIF slides. A machine learning model was employed to measure the effect of these metrics on achieving a response to ICB (SD, PR, or CR). A higher density of B lymphocytes (CD20+) was found in responders compared to non-responders to ICB (p=0.022). A positive correlation was observed between mIF and pathologist identification of TLS (R2= 0.66, p-value= <0.0001). TLS trended toward being more prevalent in responders to ICB (p=0.0906). The presence of TLS within 100 um of the tumor was associated with improved overall (p=0.04) and progression-free survival (p=0.03). A multivariate machine learning model identified TLS density as a leading predictor of response to ICB with 80% accuracy. Immune cell densities and TLS spatial location within the tumor microenvironment play a critical role in the immune response to HNSCC and may potentially outperform CPS as a predictor of ICB response.

Quantification of macrophage activity in knee synovial tissue using [18F]FEPPA positron emission tomography

ObjectiveInflammation in knee osteoarthritis (OA) is mediated primarily by synovial tissue macrophages, but non-invasive measurement of macrophage activity in vivo is a challenge. Activated macrophages markedly increase expression of the translocator protein (TSPO). In the context of other diseases TSPO-PET using the [18F]FEPPA tracer (binding to TSPO) has been reported to be an effective method for imaging macrophages in vivo. The goal of this study was to validate the use of [18F]FEPPA PET radiotracer to accurately measure macrophage activation in knee synovial tissue. DesignTen participants with late-stage OA scheduled for knee replacement surgery were recruited. Prior to surgery, 5 of the participants underwent a clinical [18F]FEPPA PET/MRI scan and the standard uptake value (SUV) in the suprapatellar synovial region was calculated. Suprapatellar synovial tissue samples were collected from all participants at the time of surgery and were imaged ex vivo with [18F]FEPPA autoradiography. Tracer uptake was compared to TSPO antibody immunostaining in serial tissue sections. The correlation between the [18F]FEPPA uptake and gold standard TSPO fluorescent intensity was measured. RESULTSThe autoradiography [18F]FEPPA signal in the synovial lining was correlated with the TSPO signal in the. lining macrophages measured by immunohistochemistry (r = 0.81, p = 0.0040, CI [0.37, 0.95]). Similarly, PET/MRI scans demonstrated similar correlation between SUV in synovial tissues in vivo and in situ TSPO signal in lining macrophages {r = 0.90, p = 0.083, CI [0.086, 0.99]) Conclusion[18F]FEPPA uptake in knee synovial tissue is strongly correlated to the expression of TSPO in synovial lining macrophages, suggesting that clinical [18F]FEPPA PET/MRI may be a valid measure of true macrophage activity in synovial tissues in knee OA. [18F]FEPPA may be an effective tool to assess macrophage activation both ex vivo and in vivo, and should be investigated further to assess its performance and measurement properties in different clinical contexts of knee OA including disease states and responses to treatment.

Gene and Protein Expression of MAGE and Associated Immune Landscape Elements in Non-Small-Cell Lung Carcinoma and Urothelial Carcinomas.

Melanoma-associated antigen-A (MAGE-A) is expressed in multiple cancers with restricted expression in normal tissue. We sought to assess the MAGE-A3/A6 expression profile as well as immune landscape in urothelial (UC) and non-small cell lung carcinoma (NSCLC). We also assessed co-expression of immune-associated markers, including programmed cell death ligand 1 (PD-L1) in tumor and/or immune cells, and assessed the effect of checkpoint inhibitor treatment on these markers in the context of urothelial carcinoma. We used formalin-fixed paraffin-embedded (FFPE) tissue sections from a variety of tumor types were screened by IHC for MAGE-A and PD-L1 expression. Gene expression analyses by RNA sequencing were performed on RNA extracted from serial tissue sections. UC tumor samples from patients treated with checkpoint inhibitors were assessed by IHC and NanoString gene expression analysis for MAGE-A and immune marker expression before and after treatment. Overall, 84 samples (57%) had any detectable MAGE-A expression. Detectable MAGE-A expression was present at similar frequencies in both tumor tissue types, with 41 (50%) NSCLC and 43 (64%) UC. MAGE-A expression was not significantly changed before and after checkpoint inhibitor therapy by both IHC and NanoString mRNA sequencing. Other immune markers were similarly unchanged post immune checkpoint inhibitor therapy. Stable expression of MAGE-A3/A6 pre and post checkpoint inhibitor treatment indicates that archival specimens harvested after checkpoint therapy are applicable to screening potential candidates for MAGE therapies.

A cross-sectional study to correlate nuclear fixative properties of formal saline and clarke’s fluid for histomorphology evaluation of human tissues

Abstract Background: Formal saline is an extensively used cheapest and easily available tissue fixative. However, formalin has been proven to have carcinogenic effects. Clarke’s fluid is an alternative to formal saline and is effective in maintaining the morphological and cellular architecture. Objectives: The objective of the study was to compare the nuclear fixative properties of Clarke’s fluid and formal saline. Materials and Methods: The fresh human tissues of thin skin, large-sized arteries, lymph nodes, spleen, and ileum were obtained from the fresh cadaver. The tissues were fixed in two different fixatives namely 10% formalin and Clarke’s fluid. Then, the tissues were histologically processed. The hematoxylin and eosin-stained serial sections of tissues were graded based on the ease of microtomy, cytoplasmic, and nuclear staining scores. Results: The nuclear properties of all the tissues immersed in Clarke’s fluid showed a median score of 3 and Grade 2 for formal saline; cytoplasmic properties for spleen and skin were median scores of Grade 3 for Clarke’s fluid. The artery was graded 3 for both the fixatives. The Kruskal–Wallis test when applied to nuclear properties, cytoplasmic adequacy, and tissue shrinkage for tissues stained with Clarke’s fluid was statistically significant (P &lt; 0.01). This inferred that Clarke’s fluid is a better nuclear fixative than formal saline. Conclusion: The imminent risks associated with formal saline paved us to explore substitute fixatives, which could provide better results and safety for laboratory workers. Clarke’s fluid is an excellent substitute for formal saline as a nuclear fixative.

Resolving multi-image spatial lipidomic responses to inhaled toxicants by machine learning.

Regional responses to inhaled toxicants are essential to understand the pathogenesis of lung disease under exposure to air pollution. We evaluated the effect of combined allergen sensitization and ozone exposure on eliciting spatial differences in lipid distribution in the mouse lung that may contribute to ozone-induced exacerbations in asthma. Lung lobes from male and female BALB/c mice were cryosectioned and acquired by high resolution mass spectrometry imaging (MSI). Processed MSI peak annotations were validated by LC-MS/MS data from scraped tissue slides and microdissected lung tissue. Images were normalized and segmented into clusters. Interestingly, segmented clusters overlapped with stained serial tissue sections, enabling statistical analysis across biological replicates for morphologically relevant lung regions. Spatially distinct lipids had higher overall degree of unsaturated fatty acids in distal lung regions compared to proximal regions. Furthermore, the airway and alveolar epithelium exhibited significantly decreased sphingolipid and glycerophospholipid abundance in females, but not in males. We demonstrate the potential role of lipid saturation in healthy lung function and highlight sex differences in regional lung lipid distribution following ozone exposure. Our study provides a framework for future MSI experiments capable of relative quantification across biological replicates and expansion to multiple sample types, including human tissue.

Increased tumor-infiltrating plasmacytoid dendritic cells express high levels of PD-L2 and affect CD8+ T lymphocyte infiltration in human laryngeal squamous cell carcinoma

The infiltration and prognostic significance of tumor-infiltrating plasmacytoid dendritic cells (TI-pDC) have been elucidated in various human solid cancers. However, the infiltrating patterns and functional importance of TI-pDC in laryngeal squamous cell carcinoma (LSCC) remain unknown. In this study, flow cytometric analyses were conducted to characterize the infiltration of dendritic cells and T lymphocytes, along with their respective subgroups in tumor tissues (TT), para-carcinoma tissues (PT), and peripheral blood (PB) from LSCC patients. Immunohistochemical staining for CD4 and CD8, as well as immunofluorescence staining for CD123, were performed on serial tissue sections to investigate the co-localization of TI-pDC and tumor-infiltrating T lymphocytes (TIL) within the tumor microenvironment (TME). Our results demonstrated significantly lower percentages of all three DC subsets in PB compared to TT and PT. Notably, the pDC percentage was markedly higher in TT than in PT. Moreover, TI-pDC percentage was significantly elevated in N+ stage patients compared to those with N0 stage. The results of survival analysis consistently demonstrated that high levels of TI-pDC infiltration were indicative of a poor prognosis. Further investigation revealed a significant negative correlation between TI-pDC and CD8+ TILs; notably, pDCs expressed an inhibitory surface molecule PD-L2 rather than PD-L1 within PT. Collectively, our findings suggest that increased TI-pDC is associated with adverse outcomes in LSCC patients while exhibiting an inhibitory phenotype that may play a crucial role in suppressing CD8+ TILs within LSCC tumors. These results highlight the potential therapeutic strategy targeting PD-L2+ pDCs for immunotherapies against LSCC.

Abstract 4779: The effects of glutamine deprivation on ovarian cancer initiation and progression

Abstract High-grade serous ovarian cancer (HGSC) is the most common subtype of ovarian cancer. Most patients with HGSC are diagnosed at advanced stage diseases. Despite the high response rate to platinum/taxane-based chemotherapy, most of these patients will develop recurrent chemoresistant disease. Recent studies showed the cancer associated fibroblasts (CAFs) in the tumor microenvironment (TME) play an important role in modulating the malignant phenotypes of HGSC. CAFs are primarily responsible for producing factors such as cytokines and growth factors to modify the TME niche to promote the malignant phenotypes of cancer cells. Recently, we have identified a previously unrecognized mechanism in which metabolism of reactive CAFs is reprogrammed through an upregulated glutamine anabolic pathway in HGSC. This dysfunctional CAF metabolism confers atypical metabolic flexibility and adaptive mechanisms in CAFs, allowing them to harness carbon and nitrogen from noncanonical sources to synthesize glutamine in nutrient-deprived conditions existing in the TME. Our preliminary data show that tumors developed from syngeneic fallopian epithelial cell-derived cancer cells injected IP into C57BL/6 mice, which started to be fed with normal diet two weeks before tumor cells injection had significantly higher tumor burden than those fed with the glutamine-deprived diet. Moreover, tumor-bearing mice treated with glutaminase inhibitor for 3 weeks demonstrated significantly lower tumor burden than those treated with the control vehicle. These findings suggest that the interplay between CAFs and HGSC cells enriches glutamine in the ovarian TME; and glutamine depletion can not only suppress ovarian tumor growth but also prevent the development of advanced progressive diseases. In addition to the direct promoting effect of glutamine on the glutamine addicted HGSC cells, increased CAF-derived glutamine can also affect the activity of other stromal cell types such as immune cells in the TME, which subsequently modulate HGSC cell growth. Imaging mass cytometry (IMC) and Mass Spectrometry Imaging (MSI) were performed on serial tissue sections of tissue microarrays generated from the abovementioned murine samples. Mice fed with a glutamine-deprived diet or treated with the glutaminase inhibitor not only have a significantly lower tumor burden but also induced dramatic changes in the cellular and molecular composition within the TME. In particular, mice fed with a glutamine-deprived diet had greater B-cell-related immune response, reduced stemness, and EMT of ovarian cancer cells, and more activated CAFs, which may result in increased stiffness of the ECM and subsequently enhance the malignant phenotype of HGSC cells. Further studies to delineate the molecular mechanisms by which spatial cellular and metabolic profiles modulate HGSC pathogenesis and progression are warranted. Citation Format: Sammy Ferri-Borgogno, Kathryn Lu, Basant T. Gamal, Chi Lam Au Yeung, Erin Seeley, Javier A. Gomez, Akshay Basi, Jared K. Burks, Olamide Animasahun, Deepak Nagrath, Samuel C. Mok. The effects of glutamine deprivation on ovarian cancer initiation and progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4779.

Resveratrol ameliorates DSS-induced ulcerative colitis by acting on mouse gut microbiota.

Ulcerative colitis (UC) is a multi-faceted, recurrent immune disorder caused by dextran sulfate sodium (DSS). The intestinal microbiota has multiple functions in the host, so UC requires long-term potent medication. The effect of resveratrol (RSV) has seldom been reported, and this study researched that. Herein, the effect of RSV and Grape seed oil that anti-inflammatory ability in experimental mice was explored, also why RSV altered Gut Microbiota has been researched. In this experiment, the effects of experimental drugs on colon length in mice with DSS-induced colitis were compared. H&E Staining was performed on serial sections of colon tissues and histological scores were determined for all groups. The expression of cyclooxygenase-2 (COX-2) and tumor necrosis factor-α (TNF-α) in the colon tissue of mice was detected by immunohistochemical staining. In the end, the α-diversity index, sobs index, and rarefaction curve of the cecal and colon microbiota of different groups of mice were measured. Bray-Curtis-based Venn diagram of PCoA (principal coordinate analysis) and OTUs distribution in mouse gut microbiota were obtained. The results showed that the use of 40mg/kg RSV (high dose) significantly reduced the severity of UC. The use of 10mg/kg RSV (low dose) significantly reduced the effect of shortened colon length in DSS mice. Compared with the DSS-treated group, the levels of COX-2 and TNF-α in the colon tissues of RSV + DSS-treated mice were significantly decreased. According to this experiment, 19 mouse gut microbiota species had a relative abundance greater than 0.1%, with Beerella, Bacteroides, Helicobacter, Oscillator, and cecum pylori being more abundant in the colon than in the colon. A higher relative abundance of Lachnospira NK4A136 was observed in DSS and RSV groups compared with the control group, whereas the opposite was observed for Alloprevotella. This proves that resveratrol increases the uniformity and diversity of gut microbes to a certain extent, and has a protective effect on the gut.

Development of the hyolaryngeal architecture in horseshoe bats: insights into the evolution of the pulse generation for laryngeal echolocation

BackgroundThe hyolaryngeal apparatus generates biosonar pulses in the laryngeally echolocating bats. The cartilage and muscles comprising the hyolarynx of laryngeally echolocating bats are morphologically modified compared to those of non-bat mammals, as represented by the hypertrophied intrinsic laryngeal muscle. Despite its crucial contribution to laryngeal echolocation, how the development of the hyolarynx in bats differs from that of other mammals is poorly documented. The genus Rhinolophus is one of the most sophisticated laryngeal echolocators, with the highest pulse frequency in bats. The present study provides the first detailed description of the three-dimensional anatomy and development of the skeleton, cartilage, muscle, and innervation patterns of the hyolaryngeal apparatus in two species of rhinolophid bats using micro-computed tomography images and serial tissue sections and compares them with those of laboratory mice. Furthermore, we measured the peak frequency of the echolocation pulse in active juvenile and adult individuals to correspond to echolocation pulses with hyolaryngeal morphology at each postnatal stage.ResultsWe found that the sagittal crests of the cricoid cartilage separated the dorsal cricoarytenoid muscle in horseshoe bats, indicating that this unique morphology may be required to reinforce the repeated closure movement of the glottis during biosonar pulse emission. We also found that the cricothyroid muscle is ventrally hypertrophied throughout ontogeny, and that the cranial laryngeal nerve has a novel branch supplying the hypertrophied region of this muscle. Our bioacoustic analyses revealed that the peak frequency shows negative allometry against skull growth, and that the volumetric growth of all laryngeal cartilages is correlated with the pulse peak frequency.ConclusionsThe unique patterns of muscle and innervation revealed in this study appear to have been obtained concomitantly with the acquisition of tracheal chambers in rhinolophids and hipposiderids, improving sound intensity during laryngeal echolocation. In addition, significant protrusion of the sagittal crest of the cricoid cartilage and the separated dorsal cricoarytenoid muscle may contribute to the sophisticated biosonar in this laryngeally echolocating lineage. Furthermore, our bioacoustic data suggested that the mineralization of these cartilages underpins the ontogeny of echolocation pulse generation. The results of the present study provide crucial insights into how the anatomy and development of the hyolaryngeal apparatus shape the acoustic diversity in bats.

JCAD deficiency attenuates activation of hepatic stellate cells and cholestatic fibrosis.

Cholestatic liver diseases including primary biliary cholangitis (PBC) are associated with active hepatic fibrogenesis, which ultimately progresses to cirrhosis. Activated hepatic stellate cells (HSCs) are the main fibrogenic effectors in response to cholangiocyte damage. JCAD regulates cell proliferation and malignant transformation in nonalcoholic steatoheaptitis-associated hepatocellular carcinoma (NASH-HCC). However, its participation in cholestatic fibrosis has not been explored yet. Serial sections of liver tissue of PBC patients were stained with immunofluorescence. Hepatic fibrosis was induced by bile duct ligation (BDL) in wild-type (WT), global JCAD knockout mice (JCAD-KO) and HSC-specific JCAD knockout mice (HSC-JCAD-KO), and evaluated by histopathology and biochemical tests. In situ-activated HSCs isolated from BDL mice were used to determine effects of JCAD on HSC activation. In consistence with staining of liver sections from PBC patients, immunofluorescent staining revealed that JCAD expression was identified in smooth muscle α-actin (α-SMA)-positive fibroblast-like cells and was significantly up-regulated in WT mice with BDL. JCAD deficiency remarkably ameliorated BDL-induced hepatic injury and fibrosis, as documented by liver hydroxyproline content, when compared to WT mice with BDL. Histopathologically, collagen deposition was dramatically reduced in both JCAD-KO and HSC-JCAD-KO mice compared to WT mice, as visualized by Trichrome staining and semi-quantitative scores. Moreover, JCAD deprivation significantly attenuated in situ HSC activation and reduced expression of fibrotic genes after BDL. JCAD deficiency effectively suppressed hepatic fibrosis induced by BDL in mice, and the underlying mechanisms are largely through suppressed Hippo-YAP signaling activity in HSCs.

Three-dimensional model of normal human dermal tissue using serial tissue sections.

Background: This study aims to construct a three-dimensional model of skin dermis utilizing continuous tissue sections, with the primary objective of obtaining anatomical structure data for normal human dermal tissues. Methods: Normal skin tissue specimens were acquired, paraffin-embedded, and subjected to HE staining. Panoramic images of skin sections were captured using a microscope. Tissue section images were aligned using the SIFT and StackReg image alignment methods, with analysis conducted using the OpenCV module. Mimics17 software facilitated the reconstruction of the skin dermal 3D model, enabling the calculation of dermal porosity and the void diameter. Results: Panoramic skin slices exhibited high-resolution differentiation of dermal fibers and cellular structures. Both SIFT and StackReg image alignment methods yielded similar results, although the SIFT method demonstrated greater robustness. Successful reconstruction of the three-dimensional dermal structure was achieved. Quantitative analysis revealed a dermal porosity of 18.96 ± 4.41% and an average pore diameter of 219.29 ± 34.27 μm. Interestingly, the porosity of the dermis exhibited a gradual increase from the papillary layer to the fourth layer, followed by a transient decrease and then a gradual increase. The distribution of the mean pore diameter mirrored the pattern observed in porosity distribution. Conclusion: Utilizing the continuous skin tissue slice reconstruction technique, this study successfully reconstructed a high-precision three-dimensional tissue structure of the skin. The quantitative analysis of dermal tissue porosity and average pore diameter provides a standardized dataset for the development of biomimetic tissue-engineered skin.

Verteporfin-Loaded Bioadhesive Nanoparticles for the Prevention of Hypertrophic Scar.

Hypertrophic scarring (HS) is a common skin injury complication with unmet needs. Verteporfin (VP) should be an ideal HS-targeted therapeutic drug due to its efficient fibrosis and angiogenesis inhibitory abilities. However, its application is restricted by its side effects such as dose-dependent cytotoxicity on normal cells. Herein, the bioadhesive nanoparticles encapsulated VP (VP/BNPs) are successfully developed to attenuate the side effects of VP and enhance its HS inhibition effects by limiting VP releasing slowly and stably in the lesion site but not diffusing easily to normal tissues. VP/BNPs displayed significant inhibition on the proliferation, migration, collagen deposition, and vessel formation of human hypertrophic scar fibroblasts (HSFBs) and dermal vascular endothelial cells (HDVECs). In a rat tail HS model, VP/BNPs treated HS exhibits dramatic scar repression with almost no side effects compared with free VP or VP-loaded non-bioadhesive nanoparticles (VP/NNPs) administration. Further immunofluorescence analysis on scar tissue serial sections validated VP/BNPs effectively inhibited the collagen deposition and angiogenesis by firmly confined in the scar tissue and persistently releasing VP targeted to nucleus Yes-associated protein (nYAP) of HSFBs and HDVECs. These findings collectively suggest that VP/BNPs can be a promising and technically advantageous agent for HS therapies.

Tryptase-Positive Mast Cells Promote Adipose Fibrosis in Secondary Lymphedema through PDGF.

Lymphedema is a chronic and progressive condition that causes physical disfigurement and psychological trauma due to the accumulation of lymphatic fluid in the interstitial space. Once it develops, lymphedema is difficult to treat because it leads to the fibrosis of adipose tissue. However, the mechanism behind this remains unclear. The purpose of this study was to investigate the involvement of mast cells (MCs) in the adipose tissues of patients with lymphedema. We found that fibrosis spread through blood vessels in the adipose tissues of lymphedema patients, and the expression of the collagen I and III genes was significantly increased compared to that of those in normal adipose tissue. Immunostaining of vimentin and α-smooth muscle actin showed that fibroblasts were the main cellular components in severely fibrotic regions. Toluidine blue staining confirmed a significant increase in the number of MCs in the adipose tissues of lymphedema patients, and immunostaining of serial sections of adipose tissue showed a significant increase in the number of tryptase-positive cells in lymphedema tissues compared with those in normal adipose tissues. Linear regression analyses revealed significant positive correlations between tryptase and the expressions of the TNF-α, platelet-derived growth factor (PDGF)-A, and PDGFR-α genes. PDGF-A-positive staining was observed in both fibroblasts and granules of tryptase-positive MCs. These results suggest that MC-derived tryptase plays a role in the fibrosis of adipose tissue due to lymphedema directly or in cooperation with other mediators.

Kyoto Collection in The Anatomical Record.

Human embryology began at the end of the 19th century and has developed using valuable human embryo specimens; the Carnegie and Blechshmidt Collections are famous examples. Although established after these two collections, the Kyoto Collection of Human Embryos and Fetuses has become the largest collection worldwide, and its major asset is 1044 serial tissue sections comprising 547 normal and 497 abnormal cases. Morphological changes have been the focus of analysis owing to the absence of fresh embryos in the Kyoto Collection. Furthermore, analysis methods have undergone significant changes. For example, morphometrics has been used to quantitatively analyze shape changes, although it may result in the loss of information on shape changes, which can hinder the visualization of analysis results. However, geometric morphometrics has been recently introduced to the fetal and embryonic stages to circumvent this problem. With the development of DNA analysis kits, several hundred DNA base pairs have been extracted from the Kyoto Collection of studies conducted from the 2000s to the 2010s through genetic analysis. Future technological advances are eagerly awaited.

Synthesis, Characterization, and Applications of a Superior Dendrimer-Based Polymer for Multiplexed Ion Beam Imaging Time-of-Flight Analysis.

High-dimensional single-cell mass spectrometric imaging techniques such as multiplexed ion beam imaging by time-of-flight mass spectrometry (MIBI-TOF), imaging mass cytometry (IMC), and flow cytometry-based CyTOF utilize antibodies conjugated to linear metal-chelating polymers. Here, we report on the synthesis and characterization of a dendrimer-based polymer and its utilization in tissue imaging using MIBI-TOF. We compared the staining performance in FFPE tissue of antibodies for lineage-specific immune proteins (CD20, CD3, CD45, FoxP3) that were conjugated with dendrimer or linear polymer. Staining of serial tissue sections with dendron-conjugated and linear-polymer-conjugated antibodies revealed comparable avidities of dendrons and linear polymers with log2 (ratio of mean positive pixel intensity of staining for linear polymers to dendrons) within the range ±0.25. Interestingly, dendron-conjugated antibodies were observed to have some advantages over linear polymer-conjugated antibodies. For example, tissue staining of a nuclear protein, FoxP3 with dendron-conjugated antibodies showed notably less background staining than that of linear-polymer-conjugated antibodies. Additionally, dendron-conjugated antibodies did not exhibit off-target cytosolic binding in neural tissue typically observed when using linear polymer conjugates. Taken together, this work provides a versatile framework for using third-generation dendron-conjugated antibodies with improved staining over conventional linear polymers.

Addressing uncertainties in correlative imaging of exogenous particles with the tissue microanatomy with synchronous imaging strategies.

Exposure to exogenous particles is of increasing concern to human health. Characterizing the concentrations, chemical species, distribution, and involvement of the stimulus with the tissue microanatomy is essential in understanding the associated biological response. However, no single imaging technique can interrogate all these features at once, which confounds and limits correlative analyses. Developments of synchronous imaging strategies, allowing multiple features to be identified simultaneously, are essential to assess spatial relationships between these key features with greater confidence. Here, we present data to first highlight complications of correlative analysis between the tissue microanatomy and elemental composition associated with imaging serial tissue sections. This is achieved by assessing both the cellular and elemental distributions in three-dimensional space using optical microscopy on serial sections and confocal X-ray fluorescence spectroscopy on bulk samples, respectively. We propose a new imaging strategy using lanthanide-tagged antibodies with X-ray fluorescence spectroscopy. Using simulations, a series of lanthanide tags were identified as candidate labels for scenarios where tissue sections are imaged. The feasibility and value of the proposed approach are shown where an exposure of Ti was identified concurrently with CD45 positive cells at sub-cellular resolutions. Significant heterogeneity in the distribution of exogenous particles and cells can be present between immediately adjacent serial sections showing a clear need of synchronous imaging methods. The proposed approach enables elemental compositions to be correlated with the tissue microanatomy in a highly multiplexed and non-destructive manner at high spatial resolutions with the opportunity for subsequent guided analysis.

The relationship between Meckel's cartilage resorption and incisor tooth germ in mice.

Our understanding of the initiation and cellular mechanisms underlying endochondral resorption of Meckel's cartilage (MC) remains limited. Several studies have shown that the resorption site of MC and the mandibular incisor tooth germ are located close to each other. However, whether incisor tooth germ development is involved in MC resorption remains unclear. In this study, we aimed to elucidate the spatio-temporal interaction between the initiation site of MC resorption and the development of incisor tooth germs in an embryonic mouse model. To this effect, we developed a histology-based three-dimensional (3D) reconstruction technique using paraffin-embedded serial sections of various tissues in the jaw. The serial sections were cut in the frontal section and the tissue constituents (e.g., MC, incisor, and mineralized mandible) were studied using conventional and enzyme-based histochemistry. The outline of each component was marked on the frontal sectional images and 3D structures were constructed. To assess the vascular architecture at the site of MC resorption, immunohistochemical staining using anti-laminin, anti-factor VIII, and anti-VEGF antibodies was performed. MC resorption was first observed on the lateral incisor-facing side of the cartilage rods at sites anterior to the mental foramen on E16.0. The 3D analysis suggested that: (a) the posterior region of the clastic cartilage resorption corresponds to the cervical loop of the incisor; (b) the cervical portion of the tooth germ inflates probably due to temporal cellular congestion prior to differentiation into matrix-producing cells; (c) the incisor tooth germ tissue is present in close proximity to MC even in mouse with continuously growing tooth and determines the disappearance of MC as the tooth development.

Abstract 3582: Differentiation of niraparib and olaparib brain penetration in a mouse brain metastatic tumor model

Abstract Purpose: There remains an unmet need to provide effective treatment therapy for patients with primary and metastatic brain tumors; lack of drug penetration across the blood brain barrier (BBB) is a key factor. Synthetic lethality remains an attractive mechanism in treating brain tumors post radiotherapy. Here, we evaluated brain penetration and distribution of niraparib and olaparib in a mouse brain tumor model. Experimental: Female mice (CrTac:NCr-Foxn1nu; 6 w/o) received 2.5E5 luciferase transfected human breast cancer line (MDA 231-BRM2-831) via intracardiac injection. Mice were imaged twice/week using bioluminescence imaging (BLI) to monitor tumor growth. On day 35, mice with brain metastases (BM) were treated via oral gavage once daily for 5 days with either niraparib (35 mg/kg, n=4 BM, n=3 control), olaparib (50 mg/kg, n=3 BM, n=3 control), or vehicle (n=3 control). Terminal blood samples and brains were collected 2 hr post final dose. Serial tissue sections were collected for MALDI-IMS, H&amp;E and IHC staining from 5 distinct horizontal planes in the brain. Tissue collected between each imaging plane was homogenized for LC-MS bioanalysis. Summary of Data: In vivo BLI imaging was used to identify mice with BM and tumor presence was confirmed ex vivo using IHC. Quantitative MALDI IMS of coronal brain sections collected from mice administered niraparib showed consistent concentrations distributed throughout the brain parenchyma with locally higher concentrations detected from tumor regions. Table 1 summarizes the LC-MS bioanalysis concentrations in plasma and bulk brain homogenates and brain section concentrations detected by MALDI IMS. The estimated mean unbound brain-to-plasma partition coefficient (Kp,uu,brain) was 3.0x and 5.6x higher for niraparib compared to olaparib in control and BM mice, respectively. Conclusions: Herein, we demonstrated that niraparib has a higher brain penetration and distribution compared to olaparib in both control mice and mice with BM. Summary of Quantification Results Displayed as Total Conc. with Adjusted Free Conc. in Parentheses Compound Group Terminal Plasma Mean (ng/mL) LC-MS Bulk Brain Homogenate Mean (ng/g) MALDI IMS Brain Section Mean (ng/g) Kp,uu Brain Niraparib Control 3847 (769) 658 (111) 532 (89) 0.18 Niraparib BM 2535 (507) 543 (91) 473 (80) 0.14 Niraparib Vehicle BLQ BLQ Olaparib Control 226 (68) 6 (2) BLQ 0.06 Olaparib BM 119 (36) 5 (2) BLQ 0.03 Olaparib Vehicle BLQ BLQ Citation Format: Mark Reid Groseclose, Jeremy Barry, Tinamarie Skedzielewski, Gerald McDermott, Chakravarthi Balabhadrapatruni, Bill Benson, Yongle Pang, David K. Lim, Hoang Tran, Mike Ringenberg, Keyur Gada, Amine Aziez, Elaine Paul, Hasan Alsaid. Differentiation of niraparib and olaparib brain penetration in a mouse brain metastatic tumor model. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3582.

Abstract 5432: Validation of spatial gene expression patterns predicted by deep convolutional neural networks from breast cancer histopathology images

Abstract Molecular profiling is central in cancer precision medicine but remains costly and is only based on tumor average profiles. Morphologic patterns observable in histopathology sections from tumors are determined by the underlying molecular phenotype and therefore have the potential to be exploited for the prediction of molecular phenotypes. Transcriptome-wide expression morphology (EMO) analysis with deep convolutional neural networks (CNN) enables the prediction of mRNA expression and proliferation markers from routine histopathology whole slide images in breast cancer. The NanoString GeoMx® Digital Spatial Profiler (DSP) platform has capabilities of associating crucial spatial information with intratumor variabilities of gene expression, serving as an ideal tool to characterize and validate the prediction of intratumor heterogeneity. The GeoMx platform also provides functionalities of overlaying and aligning hematoxylin and eosin (H&amp;E) whole slide scans on serial tissue sections with morphology marker staining to facilitate region of interest (ROI) selection to match relevant image tiles from the model input, which is a key step to ensure accuracy for the evaluation of the model predictions. The GeoMx RNA Immune Pathways Panel contains 84 gene targets including key genes involved in immune pathways and tumorigenesis and was used to validate the prediction of mRNA expression output from deep CNN models. Citation Format: Kathy Ton, Yinxi Wang, Liuliu Pan, Kimmo Kartasalo, Balazs Acs, Philippe Weitz, Liang Zhang, Yan Liang, Johan Hartman, Masi Volkonen, Christer Larsson, Pekka Ruusuvuori, Joseph Beechem, Mattias Rantalainen. Validation of spatial gene expression patterns predicted by deep convolutional neural networks from breast cancer histopathology images. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5432.