Bright-field Microscopy Research Articles

Use of artificial intelligence for the detection of infections caused by Candida spp.: a systematic review

Introduction: Candida spp. is composed of more than 160 species present in nature. However, only about 20 of them pose a threat to humans today. Situations of imbalance can lead to the spread and, consequently, fungal infection in various regions of the body. In this sense, Artificial Intelligence (AI) has proven to be an important tool in the identification of these infectious conditions. This study aims to evaluate the main studies related to the use of AI in the identification of fungi of the genus Candida and/or in the diagnosis of candidemia. Methods: Systematic review was conducted in the Pubmed, Lilacs, Cochrane and Embase databases, according to the guidelines of the International Preferred Reporting Items for Systematic Review and Meta-Analyses (PRISMA). Results: Of the 403 studies identified, 4 were selected for inclusion in the review. Each study employed different means to obtain data, including an image bank of clinical specimens captured by brightfield microscopy, Raman spectroscopy, E-Nose, and clinical and laboratory databases. Subsequently, these data were analyzed by AI's, highlighting InceptionV3, 1-D CNN, Inception Time and Random Forest, which obtained the best performances. Conclusion: From the analysis of the studies, it was noted that the machine learning tools ensured greater agility and cost reduction, compared to traditional methods, in the detection of Candida spp. infection. In addition, these techniques demonstrated higher than average accuracy, sensitivity, and specificity. Thus, it is evident that AI's are technologies of great potential for the identification of fungal infections by Candida species.

A Binocular Color Line-Scanning Stereo Vision System for Heavy Rail Surface Detection and Correction Method of Motion Distortion.

Thanks to the line-scanning camera, the measurement method based on line-scanning stereo vision has high optical accuracy, data transmission efficiency, and a wide field of vision. It is more suitable for continuous operation and high-speed transmission of industrial product detection sites. However, the one-dimensional imaging characteristics of the line-scanning camera cause motion distortion during image data acquisition, which directly affects the accuracy of detection. Effectively reducing the influence of motion distortion is the primary problem to ensure detection accuracy. To obtain the two-dimensional color image and three-dimensional contour data of the heavy rail surface at the same time, a binocular color line-scanning stereo vision system is designed to collect the heavy rail surface data combined with the bright field illumination of the symmetrical linear light source. Aiming at the image motion distortion caused by system installation error and collaborative acquisition frame rate mismatch, this paper uses the checkerboard target and two-step cubature Kalman filter algorithm to solve the nonlinear parameters in the motion distortion model, estimate the real motion, and correct the image information. The experiments show that the accuracy of the data contained in the image is improved by 57.3% after correction.

Multimodal 2D and 3D microscopic mapping of growth cartilage by computational imaging techniques – a short review including new research

Being able to image the microstructure of growth cartilage is important for understanding the onset and progression of diseases such as osteochondrosis and osteoarthritis, as well as for developing new treatments and implants. Studies of cartilage using conventional optical brightfield microscopy rely heavily on histological staining, where the added chemicals provide tissue-specific colours. Other microscopy contrast mechanisms include polarization, phase- and scattering contrast, enabling non-stained or ‘label-free’ imaging that significantly simplifies the sample preparation, thereby also reducing the risk of artefacts. Traditional high-performance microscopes tend to be both bulky and expensive. Computational imaging denotes a range of techniques where computers with dedicated algorithms are used as an integral part of the image formation process. Computational imaging offers many advantages like 3D measurements, aberration correction and quantitative phase contrast, often combined with comparably cheap and compact hardware. X-ray microscopy is also progressing rapidly, in certain ways trailing the development of optical microscopy. In this study, we first briefly review the structures of growth cartilage and relevant microscopy characterization techniques, with an emphasis on Fourier ptychographic microscopy (FPM) and advanced x-ray microscopies. We next demonstrate with our own results computational imaging through FPM and compare the images with hematoxylin eosin and saffron (HES)-stained histology. Zernike phase contrast, and the nonlinear optical microscopy techniques of second harmonic generation (SHG) and two-photon excitation fluorescence (TPEF) are explored. Furthermore, X-ray attenuation-, phase- and diffraction-contrast computed tomography (CT) images of the very same sample are presented for comparisons. Future perspectives on the links to artificial intelligence, dynamic studies and in vivo possibilities conclude the article.

Electrowriting of SU-8 Microfibers.

As microfiber-based additive manufacturing (AM) technologies, melt electrowriting (MEW) and solution electrowriting (SEW) have demonstrated efficacy with more biomedically relevant materials. By processing SU-8 resin using MEW and SEW techniques, a material with substantially different mechanical, thermal, and optical properties than that typically processed is introduced. SU-8 polymer is temperature sensitive and requires the devising of a specific heating protocol to be properly processed. Smooth-surfaced microfibers resulted from MEW of SU8 for a short period (from 30 to 90 min), which provides the greatest control and, thus, reproducibility of the printed microfibers. This investigation explores various parameters influencing the electrowriting process, printing conditions, and post-processing to optimize the fabrication of intricate 3D structures. This work demonstrates the controlled generation of straight filaments and complex multi-layered architectures, which were characterized by brightfield, darkfield, and scanning electron microscopy (SEM). This research opens new avenues for the design and development of 3D-printed photonic systems by leveraging the properties of SU-8 after both MEW and SEW processing.

Fast, Easy, and Comprehensive Techniques for Microscopic Observations of Fungal and Oomycete Organisms Inside the Roots of Herbaceous and Woody Plants.

The roots of herbaceous and woody plants growing in soil are complex structures that are affected by both natural and artificial fungal colonization to various extents. To obtain comprehensive information about the overall distribution of fungi or oomycetes inside a plant root system, rapid, effective, and reliable screening methods are required. To observe both fine roots, i.e., a common site for penetration of fungi and oomycetes, and mature roots, different techniques are required to overcome visual barriers, such as root browning or tissue thickening. In our protocol, we propose using fast, cost-effective, and non-harmful methods to localize fungal or oomycete structures inside plant roots. Root staining with a fluorescent dye provides a quick initial indication of the presence of fungal structures on the root surfaces. The protocol is followed by clearing and staining steps, resulting in a deeper insight into the root tissue positioning, abundance, and characteristic morphological/reproductive features of fungal or oomycete organisms. If required, the stained samples can be prepared by using freeze-drying for further observations, including advanced microscopic techniques. Key features • The protocol enhances tissue-clearing techniques employing KOH or NaOH and is applicable to a broad range of roots from different plant species. • Hydroxides are mixed with hydrogen peroxide to obtain an efficient bleaching solution, which effectively clears roots without causing significant tissue damage. • The protocol could also be used for staining of fungi or oomycetes localized both on the root surface or inside the root tissues. • Simple combination of non-fluorescent methyl blue and fluorescent solophenyl flavine dyes allows the observation of fungal organisms in both brightfield and fluorescence microscopy.

Effects of temporal IFNγ exposure on macrophage phenotype and secretory profile: exploring GMP-Compliant production of a novel subtype of regulatory macrophages (MregIFNγ0) for potential cell therapeutic applications

BackgroundMacrophages are involved in tissue homeostasis, angiogenesis and immunomodulation. Proangiogenic and anti-inflammatory macrophages (regulatory macrophages, Mreg) can be differentiated in-vitro from CD14+ monocytes by using a defined cell culture medium and a stimulus of IFNγ.Aim of the studyTo scrutinize the potential impact of temporal IFNγ exposure on macrophage differentiation as such exposure may lead to the emergence of a distinct and novel macrophage subtype.MethodsDifferentiation of human CD14+ monocytes to Mreg was performed using a GMP compliant protocol and administration of IFNγ on day 6. Monocytes from the same donor were in parallel differentiated to MregIFNγ0 using the identical protocol but with administration of IFNγ on day 0. Cell characterization was performed using brightfield microscopy, automated and metabolic cell analysis, transmission electron microscopy, flow cytometry, qPCR and secretome profiling.ResultsMreg and MregIFNγ0 showed no differences in cell size and volume. However, phenotypically MregIFNγ0 exhibited fewer intracellular vesicles/vacuoles but larger pseudopodia-like extensions. MregIFNγ0 revealed reduced expression of IDO and PD-L1 (P < 0.01 for both). They were positive for CD80, CD14, CD16 and CD38 (P < 0.0001vs. Mreg for all), while the majority of MregIFNγ0 did not express CD206, CD56, and CD103 on their cell surface (P < 0.01 vs. Mreg for all). In terms of their secretomes, MregIFNγ0 differed significantly from Mreg. MregIFNγ0 media exhibited reduced levels of ENA-78, Osteopontin and Serpin E1, while the amounts of MIG (CXCL9) and IP10 were increased.ConclusionExposing CD14+ monocytes to an alternatively timed IFNγ stimulation results in a novel macrophage subtype which possess additional M1-like features (MregIFNγ0). MregIFNγ0 may therefore have the potential to serve as cellular therapeutics for clinical applications beyond those covered by M2-like Mreg, including immunomodulation and tumor treatment.

Ultra-high electrostrain in flux-grown (Bi0.5Na0.5)TiO3-0.06BaTiO3-0.03(K0.5Na0.5)NbO3 single crystals around the nonergodic/ergodic crossover region

In this study, we successfully grew large-sized single crystals (12 × 11 × 8 mm³) of 0.91Bi0.5Na0.5TiO3-0.06BaTiO3-0.03(K0.5Na0.5)NbO3 (BNT-6BT-3KNN) with a <100>c orientation using the flux growth method. This marks the first instance of achieving such crystals. The single crystals display double hysteresis loops, featuring a maximum polarization of ∼64.6 μC/cm2. Capitalizing on the nonergodic/ergodic boundary, the single crystals exhibit a substantial strain (Sm) of 0.91% at the maximum electric field of 20 kV/cm and an exceptionally high inverse piezoelectric coefficient d33* of 4550 pm/V. The creation of long-range domains from needle-like nanodomains and polar-nano regions under the application of an electric field was confirmed through bright-field transmission electron microscopy and piezoresponse force microscopy analyses. The phase diagram for BNT-6BT-3KNN single crystals was constructed by analyzing the temperature dependence of dielectric, ferroelectric, and electrostrain behaviors.

Mini Winnies: scaled down and transparent Winogradsky columns for microscopy in microbiology education.

Winogradsky columns were invented by Sergei Winogradsky in the 1880s and have commonly been used as a microbiology classroom learning tool in K-12 and collegiate education. However, they can be challenging to examine with microscopy. We scaled down Winogradsky columns into nuclear magnetic resonance (NMR) tubes and replaced the natural sediment with a transparent soil substitute toward the goal of observing the microbial growth under a bright-field microscope without column disassembly. Using this "Mini Winnie" approach, students can practice their microscopy skills while observing microbial growth inside the column after only days of incubation on the laboratory windowsill. Overall, we believe that the Mini Winnies provide a simple method for maximizing student engagement while giving them a greater understanding of how microorganisms interact in the environment.

Novel Chromogens for Immunohistochemistry in Spatial Biology.

Spatial relations between tumor cells and host-infiltrating cells are increasingly important in both basic science and clinical research. In this study, we have tested the feasibility of using standard methods of immunohistochemistry (IHC) in a multiplex staining system using a newly developed set of chromogenic substrates for the peroxidase and alkaline phosphatase enzymes. Using this approach, we have developed a set of chromogens characterized by (1) providing fine cellular detail, (2) non-overlapping spectral profiles, (3) an absence of interactions between chromogens, (4) stability when stored, and (5) compatibility with current standard immunohistochemistry practices. When viewed microscopically under brightfield illumination, the chromogens yielded the following colors: red, black, blue, yellow, brown, and green. By selecting compatible color combinations, we have shown feasibility for four-color multiplex staining. Depending on the particular type of analysis being performed, visual analysis, without the aid of computer-assisted image analysis, was sufficient to differentiate up to four different markers.

Neuropathological findings in Down syndrome, Alzheimer's disease and control patients with and without SARS-COV-2: preliminary findings.

The SARS-CoV-2 virus that led to COVID-19 is associated with significant and long-lasting neurologic symptoms in many patients, with an increased mortality risk for people with Alzheimer's disease (AD) and/or Down syndrome (DS). However, few studies have evaluated the neuropathological and inflammatory sequelae in postmortem brain tissue obtained from AD and people with DS with severe SARS-CoV-2 infections. We examined tau, beta-amyloid (Aβ), inflammatory markers and SARS-CoV-2 nucleoprotein in DS, AD, and healthy non-demented controls with COVID-19 and compared withnon-infected brain tissue from each diseasegroup (total n = 24). A nested ANOVA was used to determine regional effects of the COVID-19 infection on arborization of astrocytes (Sholl analysis) and percent-stained area of Iba-1 and TMEM 119. SARS-CoV-2 antibodies labeled neurons and glial cells in the frontal cortex of all subjects with COVID-19, and in the hippocampus of two of the three DS COVID-19 cases. SARS-CoV-2-related alterations were observed in peri-vascular astrocytes and microglial cells in the gray matter of the frontal cortex, hippocampus, and para-hippocampal gyrus. Bright field microscopy revealed scattered intracellular and diffuse extracellular Aβ deposits in the hippocampus of controls with confirmed SARS-CoV-2 infections. Overall, the present preliminary findings suggest that SARS-CoV-2 infections induce abnormal inflammatory responses in Down syndrome.

Engineering Long‐Releasing Hollow‐like or Condensed Progesterone Hormone Microcrystals with Controlled Polymorphism

Progesterone is an endogenous steroid hormone involved in the menstrual cycle, pregnancy, and embryogenesis of humans and other species. Progesterone crystallization techniques have previously reported. Among these techniques, solvent crystallization and different solvent:anti‐solvent systems are considered. Herein, the selective development of either hollow‐like or condensed progesterone microcrystals in elevated yield with controlled polymorphism, habit, and release is described for the first time. For the hollow microcrystals, isopropyl alcohol (IPA) and double‐deionized water (DDW) system is developed as solvent:anti‐solvent, while acetonitrile (AcN) and DDW system are developed for the condensed microcrystals. The microcrystals obtained from both developed crystallization systems are thoroughly investigated with varied microscopic techniques, including brightfield and scanning electron microscopy (SEM), thermal analysis by differential scanning calorimetry (DSC), and crystallography by powder X‐ray diffraction (PXRD) and single XRD, and have been compared. Results show that the crystals of the IPA:DDW crystallization system are hollow and exhibit several habits, whereas the microcrystals of the AcN:DDW crystallization system are more condensed. However, both systems are found to have a wide crystal size distribution of one stable polymorph and are thus highly useful for tunable release. More importantly, these microcrystals exhibit elongated and slow release for 14 days under an expedited release conditions model, indicating suitability for long‐term and potential localized release applications.

Exploring aptamers for targeted enrichment of X sperm in bovine: unraveling selective potential

Nucleic acid aptamers have been used in the past for the development of diagnostic methods against a number of targets such as bacteria, pesticides, cancer cells etc. In the present study, six rounds of Cell-SELEX were performed on a ssDNA aptamer library against X-enriched sperm cells from Sahiwal breed cattle. Sequencing was used to examine the aptamer sequences that shown affinity for sperm carrying the X chromosome in order to find any possible X-sperm-specific sequences. Out of 35 identified sequences, 14 were selected based on bioinformatics analysis like G-Score and Mfold structures. Further validation of their specificity was done via fluorescence microscopy. The interaction of biotinylated-aptamer with sperm was also determined by visualizing the binding of streptavidin coated magnetic beads on the head region of the sperm under bright field microscopy. Finally, a real-time experiment was designed for the validation of X-sperm enrichment by synthesized aptamer sequences. Among the studied sequences, aptamer 29a exhibited a higher affinity for X sperm compared to Y sperm in a mixed population of sperm cells. By using aptamer sequence 29a, we obtained an enrichment of 70% for X chromosome bearing sperm cells.

A large and diverse brain organoid dataset of 1,400 cross-laboratory images of 64 trackable brain organoids

Brain organoids represent a useful tool for modeling of neurodevelopmental disorders and can recapitulate brain volume alterations such as microcephaly. To monitor organoid growth, brightfield microscopy images are frequently used and evaluated manually which is time-consuming and prone to observer-bias. Recent software applications for organoid evaluation address this issue using classical or AI-based methods. These pipelines have distinct strengths and weaknesses that are not evident to external observers. We provide a dataset of more than 1,400 images of 64 trackable brain organoids from four clones differentiated from healthy and diseased patients. This dataset is especially powerful to test and compare organoid analysis pipelines because of (1) trackable organoids (2) frequent imaging during development (3) clone diversity (4) distinct clone development (5) cross sample imaging by two different labs (6) common imaging distractors, and (6) pixel-level ground truth organoid annotations. Therefore, this dataset allows to perform differentiated analyses to delineate strengths, weaknesses, and generalizability of automated organoid analysis pipelines as well as analysis of clone diversity and similarity.

A facile microwave assisted synthesis of thymohydroquinone with antibiofilm properties against Salmonella typhi and Listeria monocytogenes

Thymoquinone (TQ), a monoterpene isolated from the Nigella sativa L. seeds using the oil expeller method, gave about 40% of the total oil. Clevenger hydrodistillation of the obtained oil yielded volatile oil (0.55%) with 30% of TQ. Subsequent purification through silica gel column chromatography resulted in the isolation of pure TQ. The microwave assisted reduction of TQ with zinc and acetic acid resulted in the formation of thymohydroquinone within 5 min (THQ, yield 85%). The conventional reduction method gives 42% - 60% yield and is found to be time-consuming (4–12 h). The prepared THQ was tested at various concentrations for the inhibition of planktonic as well as biofilm of Listeria monocytogenes Scott A and Salmonella typhi FB231 bacterial cultures. Minimum inhibitory concentration (MIC) using MTT assay showed 50 μg/mL and 100 μg/mL for L. monocytogenes and S. typhi, respectively. Further, the biofilm growth inhibition and development were assessed using a crystal violet (CV) assay, which confirmed that THQ and TQ significantly eradicated biofilm formed after 6 h and 24 h, as evidenced by bright field microscopy. Thus, the results provide scope on the quicker microwave assisted synthesis, which is considered a greener method and the THQ showed potential antimicrobial and antibiofilm activities against common food borne pathogens.

Near‐Infrared Metalens Empowered Dual‐Mode High Resolution and Large FOV Microscope

AbstractThe spiral phase contrast microscope can clearly distinguish the morphological information of the low contrast objects (i.e., biological samples) owing to the isotropic edge‐enhancement effect, while the bright field microscope can image the overall morphology of amplitude objects. However, the imaging resolution, magnification, and field of view of conventional spiral phase contrast microscopes based on 4f filtering configuration are limited by the system's complexity. Here, compact dual‐mode microscopes working at near‐infrared using the engineered metalens are reported, which can be tuned between the spiral phase contrast imaging and bright field imaging by polarization control. The metalens combine the high‐resolution objective lens and polarization‐controlled phase filter into a single‐layer nanofins array. Two infinity‐corrected microscope systems are demonstrated to achieve subwavelength resolution (0.7λ), large magnification (58X), and large field of view (600 × 800 µm). Unstained onion epidermal is imaged by the microscope to show the dual‐mode imaging ability for the biological sample. Finally, a singlet dual‐mode microscope system is demonstrated to show the edge‐detection application for industrial standards. These results can open new opportunities in applications of biological imaging, industrial machine vision, and semiconductor inspection.

Diving head-first into brain intravital microscopy.

Tissue microenvironments during physiology and pathology are highly complex, meaning dynamic cellular activities and their interactions cannot be accurately modelled ex vivo or in vitro. In particular, tissue-specific resident cells which may function and behave differently after isolation and the heterogenous vascular beds in various organs highlight the importance of observing such processes in real-time in vivo. This challenge gave rise to intravital microscopy (IVM), which was discovered over two centuries ago. From the very early techniques of low-optical resolution brightfield microscopy, limited to transparent tissues, IVM techniques have significantly evolved in recent years. Combined with improved animal surgical preparations, modern IVM technologies have achieved significantly higher speed of image acquisition and enhanced image resolution which allow for the visualisation of biological activities within a wider variety of tissue beds. These advancements have dramatically expanded our understanding in cell migration and function, especially in organs which are not easily accessible, such as the brain. In this review, we will discuss the application of rodent IVM in neurobiology in health and disease. In particular, we will outline the capability and limitations of emerging technologies, including photoacoustic, two- and three-photon imaging for brain IVM. In addition, we will discuss the use of these technologies in the context of neuroinflammation.

Single-Cell Sensitive Colorimetric pH Detection Based on Microscope Ratiometric Grayscale.

Rapid and accurate identification of the intracellular pH is critical in the field of biomedicine. In this work, we effectively identified and quantified the intracellular pH and its distribution at the single-cell level using an image sensor based on an ordinary bright-field optical microscope that divided the cell staining images into their red (R) and blue (B) channels. The grayscale of the R and B channels was subjected to a ratiometric operation to generate ratiometric grayscale cell images of the microscope. A standard curve of pH against ratiometric grayscale curve was then obtained by incubating HeLa cells at pH 6.00-7.60 in a high concentration K+ ion buffer solution containing nigericin for obtaining certain intracellular pH values. A good correlation was evidenced between pH and the ratiometric grayscale of the R and B channels in the pH range of 6.00-7.60. Subsequently, the intracellular pH value of the A549 cells under the experimental conditions was measured to be 7.22 ± 0.01 by the method. Furthermore, the changes in the intracellular pH of HeLa cells stimulated with hydrogen peroxide were sensitively monitored, which demonstrated the applicability of the method. Due to its ease of use, the developed colorimetric microscopy pH detection and monitoring method provide prospects for pH-related single-cell studies.

285 Impact of feeding strategies on semen concentration, motility, and morphology of the mature AI boar

Abstract The objective of this experiment was to determine if varying feeding strategies of maintenance requirements of boars impacts semen concentration, motility, morphology, and cortisol of the working boar. This experiment was conducted at Purdue University over two 9-wk periods with 1-wk before each 9-wk period serving as baseline for analysis. Boars (n = 28; Acuity and Topig Norvsin) from two age groups (9 and 15 mo) and two genetics (maternal and terminal) were used. Boars were blocked by breed and age and randomly allocated to receive one of three feed intake levels of a corn-soybean meal-based diet formulated to meet NRC (2012) maintenance requirements: HIGH (150% maintenance, n = 9); MAIN (100% maintenance, n = 10); and LOW (80% maintenance, n = 9) during the first 9-wk period. During the second 9-wk period remaining HIGH and LOW boars were crossed-over to opposite treatments (HIGH-LOW, n = 9; LOW-HIGH, n = 7), and maintenance boars were unchanged (MAIN-MAIN, n = 8). Semen was collected once/wk using the gloved hand method and 3 mL of ejaculate was diluted in 27 mL of extender (Androhep Plus, Minitube). Semen concentration (Nucleocounter SP-100), motility (Computer Assisted Sperm Analysis, CASA, CEROS II), and sperm cell morphology (phase-contrast, bright-field microscopy) were analyzed. Data were analyzed using PROC MIXED in SAS 9.4 with boar as experimental unit with main effects of feed intake and age with phase (3-wk period) as repeated measure. Significance was determined at P &amp;lt; 0.05 and a trend 0.05 &amp;lt; P ≤ 0.10. Three-way interactions for the first 9-wk period of feed intake*age*phase was observed for total sperm (P &amp;lt; 0.10) and total normal sperm (P &amp;lt; 0.05) with old boars fed HIGH treatment increasing sperm production, but old boars fed MAIN and LOW decreasing sperm production while young boars had minimal changes in sperm production regardless of feeding levels in the first 9-wk period. No other interactions or main effects were observed for the first period. During the second 9-wk period two-way interactions were observed for total sperm (FI*Age; P &amp;lt; 0.05) and total normal sperm (FI*Age; P &amp;lt; 0.01) with old LOW-HIGH boars having the highest total sperm and total normal sperm concentrations and young LOW-HIGH boars having the least total sperm and total normal sperm concentrations. HIGH-LOW fed boars had the greatest decrease in semen volume compared with the other treatments (FI*Phase; P &amp;lt; 0.01). Boars fed MAIN had minimal impacts on semen parameters compared with HIGH and LOW feed intake treatments. HIGH and LOW feed intake was observed to impact semen parameters in old boars faster than young boars with neither age being able to return to their baseline semen parameters observed at the start of the trial. Future research is needed with varying ages and feed intakes to better understand how nutritional strategies impact semen quantity and quality in the boar stud.

Abstract PO4-01-08: Co-expression of estrogen receptor (ER), progesterone receptor (PR), and Ki67 in a single breast cancer cell indicates a favorable prognosis in ER-positive breast cancer

Abstract Background: ER-positive breast cancer is biologically and clinically divided into two subtypes: luminal-A and luminal-B. This classification is mainly based on the status of cellular proliferation. At least two different pathways drive cellular proliferation in ER-positive breast cancer. One is a classical pathway where ER binds to estrogen responsive element (ERE), which leads to the expression of downstream molecules, including PR. The other is a non-classical pathway where a complex of ER and relevant factors bind to different sites than ERE. Growth factor signaling has been suggested to potentiate a non-classical pathway. We hypothesized that examining PR status in ER-positive proliferating cells could tell which pathway is more dominant in ER-positive breast cancer. Methods: To test the hypothesis, we used a newly developed triplex immunohistochemistry (IHC) that detects three molecules simultaneously under bright-field microscopy. Postmenopausal patients who were treated with neoadjuvant endocrine therapy with aromatase inhibitors from January 2007 to September 2016 at Saitama Cancer Center were included in this study. ER, PR, and Ki67 expressions were assessed in a single slide using the triplex IHC assay with anti-ER antibody (clone SP1), anti-PR antibody (clone 1E2), and anti-Ki67 antibody (clone 30-9). ER, PR and Ki67 expression was assessed in a single cell nucleus of cancer cells (567 to 4871 cells) from multiple areas in each case. An ER-positive proliferating cell was defined as an ER-positive and Ki67-positive cell. PR status in ER-positive proliferating cells was assessed. When PR was expressed in more than 50% of ER-positive proliferating cells in a clinical case, the tumor was categorized as the PR-positive group. Luminal A and luminal B breast cancers were defined based on the pre-treatment Ki67 labeling index with a cut-off of 14 %. Statistical analyses included the Mann-Whitney test, the log-rank test, and the Cox proportional hazard model. Results: Pre-treatment tissues from 55 patients were evaluated. The median age was 62 (range: 54 to 80) years. The patients were grouped into PR-positive and PR-negative groups. The two groups had no differences in age and pre-treatment T and N stages. The median pre-treatment Ki67 labeling index was 5.9 % in the PR-positive group and 9.9 % in the PR-negative group, which showed a statistically significant difference (P = 0.01). Clinical response to neoadjuvant endocrine therapy was compared, and no difference was observed. The median post-treatment Ki67 labeling index was 3.6 % in the PR-positive group and 13.1 % in the PR-negative group with a significant difference (P = 0.035). The survival was compared between the two groups. The PR-positive group showed a significantly more favorable disease-free survival (DFS) than the PR-negative group (P = 0.0079). To adjust for the background differences including Ki67 and PR status, a multivariate analysis was performed and showed that the PR-positive group had a significantly better DFS than the PR-negative group independent of clinical stage, Ki67 labeling index, and PR status (P = 0.042). Breast cancer-specific survival (BCSS) was also better in the PR-positive group than in the PR-negative group after adjusting for clinical stage, Ki67 labeling index, and PR status (P = 0.043). Interestingly, among patients with luminal A tumors, those in the PR-positive group showed a better DFS than those in the PR-negative group (P = 0.022). Conclusion: PR status in ER-positive proliferating cells was an independent prognostic factor in DFS and BCSS and divided patients with luminal A tumors further into two prognostic groups. Citation Format: Takayuki Ueno, Rie Horii, Hiroshi Matsumoto, Makiko Ono, Yurina Maeshima, Hiroaki Nitta. Co-expression of estrogen receptor (ER), progesterone receptor (PR), and Ki67 in a single breast cancer cell indicates a favorable prognosis in ER-positive breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-01-08.

Abstract PO3-07-01: Multiscale Computational Analysis of Patient-Matched Multi-Modal Imaging of Breast Tissue Microenvironment

Abstract Introduction: Mammographic breast density is a strong quantitative cancer risk factor, but its link to underlying tissue and cellular level changes are weakly understood. Understanding the complex restructuring changes in the breast tumor microenvironment and its impact on breast density is integral to improving risk assessment and treatment strategies. A novel approach was developed to stratify mammographic dense breast tissue into areas of active dense tissue and passive dense tissue. Active dense tissue is structurally reorganizing and links to cancer dynamics, whereas passive dense mammographic tissue remains organized.1,2 A complementary wavelet-based analysis technique to measure the multiscale anisotropy of collagen fibers from Second-Harmonic Generation (SHG) imaging demonstrated clinical cancer diagnostic potential in mouse carcinoma and human pancreatic cancer.3,4 This tool is now leveraged for a multi-modal study in which patient-matched H&amp;E tissue biopsies are imaged using both bright-field microscopy and SHG imaging to compare with the patient’s corresponding mammograms. Stratifying the mammographic density into active and passive dense areas, a correlation between biopsy tissue anisotropy and mammographic density subtypes can be calculated to help understand how tumor microenvironment changes affect a patient’s mammographic breast tissue composition. Methods: SHG and brightfield-imaged biopsy slides from 10 patients (five malignant, five benign) collected from Maine Medical Center (Maine, US) were analyzed using the 2D Wavelet Transform Modulus Maxima (WTMM) Anisotropy Method generating a large-scale and a small-scale anisotropy factor as well as a scale-combined anisotropy calculated from their large- and small-scale anisotropy separation. A multi-modal anisotropy score was calculated by adding the imaged area’s H&amp;E combined anisotropy score with its respective SHG combined anisotropy score. This analysis was done on eight randomly selected ductal areas for each imaging modality on a slide and correlated with the measured areas of density subtypes from the whole mammographic view. Linear regression was used for determining correlation between H&amp;E and SHG anisotropy factors as well as with active and passive mammographic density subtype measurements. Results: The malignant patients’ small-scale H&amp;E anisotropy factor positively correlated with their small scale SHG anisotropy factor (R2=0.571). No correlation was found for the benign patients (R2=0.011). The strongest correlations were found between benign patients’ mammographic passive (positive correlation) and active (negative correlation) dense tissues vs the H&amp;E anisotropy scale-combined factor (R2=0.858 and R2=0.961, respectively). The largest malignant patient correlation was a negative relationship between SHG anisotropy scale-combined factor and overall mammographic density (inclusive of both active and passive dense tissue) (R2=0.891). The multi-modal anisotropy score correlated with overall mammographic density for both benign (positive correlation, R2=0.698) and malignant patients (negative correlation, R2=0.773). Combining the multi-modal anisotropy factor from SHG and H&amp;E with the active dense tissue measurement using K-Means clustering, 5/5 malignant and 4/5 benign patients were correctly classified. Conclusion: Density in mammograms correlates with underlying quantitative changes in a patient’s tissue and collagen anisotropy. Combining both H&amp;E and SHG analysis on tissue biopsy with active and passive density metrics from computational mammography leads to the best predictive potential than any one method on their own.