Stromal Cell Counts Research Articles

CD25+FOXP3+CD45RA− regulatory T-cell infiltration as a prognostic biomarker for endometrial carcinoma

BackgroundRegulatory T (Treg) cells reportedly play crucial roles in tumor angiogenesis as well as antitumor immunity. In order to explore their therapeutic potential, we investigated the precise prognostic impact of Treg markers in endometrial carcinoma.MethodsWe performed multiplexed immunofluorescence and quantitative image analyses of CD25, FOXP3, CTLA4, and CD45RA in tumor specimens from 176 consecutive patients treated at our institution for primary endometrial carcinomas. Bioinformatics analyses were further conducted to corroborate the findings.ResultsHigh CD25+, FOXP3+, and CD25+FOXP3+CD45RA− stromal cell counts correlated with better overall survival (OS) (p = 0.00019, 0.028 and 0.0012) and MSI-high (p = 0.015, 0.016 and 0.047). High CD45RA+ stromal cell count was associated with superficial myometrial invasion (p = 0.0038). Bioinformatics survival analysis by Kaplan-Meier plotter showed that high CD25, FOXP3, CTLA4, and CD45RA mRNA expressions correlated with better OS (p = 0.046, 0.00042, 0.000044, and 0.0022). Univariate and multivariate analyses with various clinicopathologic prognostic factors indicated that high CD25+ or CD25+FOXP3+CD45RA− stromal cell count was significant and independent for favorable OS (p = 0.0053 and 0.0015). We subsequently analyzed the correlations between the multiplexed immunofluorescence results and treatment-free interval (TFI) after primary chemotherapy in recurrent cases, finding no significant associations. Further analysis revealed that high ratio of CD25+ : CD8+ cell count or CD25+FOXP3+CD45RA− : CD8+ cell count correlated with longer TFI (p = 0.021 and 0.021).ConclusionThe current observations suggest that the balance between CD25+ or CD25+FOXP3+CD45RA− cells and CD8+ cells, corresponding to promoting or inhibiting effect on tumor angiogenesis, affect tumor chemosensitivity leading to prognostic significance. CD25+FOXP3+CD45RA− effector Treg tumor infiltration may serve as a useful prognostic biomarker and a potential target for immunotherapeutic manipulation of tumor chemosensitivity by novel management for advanced/recurrent endometrial carcinomas.

Sex bias in utero alters ovarian reserve but not uterine capacity in female offspring†.

Environmental stressors to which a fetus is exposed affect a range of physiological functions in postnatal offspring. We aimed to determine the in utero effect of steroid hormones on the reproductive potential of female offspring using a porcine model. Reproductive tracts of pigs from female-biased (>65% female, n = 15), non-biased (45-54.9% female, n = 15), and male-biased litters (<35% females, n = 9) were collected at slaughter (95-115kg). Ovaries and uterine horns were processed for H&E or immunohistochemistry. Variability of data within groups was analyzed with a Levene's test, while data were analyzed using mixed linear models in R. In the ovarian reserve, there was a significant birth weight by sex ratio interaction (P = 0.015), with low birth weight pigs from male-biased litters having higher numbers of primordial follicles with opposite trends seen in pigs from female-biased litters. Sex bias held no effect on endometrial gland development. A lower birth weight decreased the proportion of glands found in the endometrium (P = 0.045) and was more variable in both male-biased and female-biased litters (P = 0.026). The variability of primordial follicles from male-biased litters was greater than non- and female-biased litters (P = 0.014). Similarly, endometrial stromal nuclei had a greater range in male- and female-biased litters than non-biased litters (P = 0.028). A crucial finding was the greater variability in primordial follicles in the ovaries from females derived from male-biased litters and stromal cell count in the endometrium of females from male- and female-biased litters. This could be inflating the variability of reproductive success seen in females from male-biased litters.

The Analgesic Efficacy of Intradiscal Injection of Bone Marrow Aspirate Concentrate and Culture-Expanded Bone Marrow Mesenchymal Stromal Cells in Discogenic Pain: A Systematic Review.

Pain originating from the intervertebral disc (discogenic pain) is a prevalent manifestation of low back pain and is often challenging to treat. Of recent interest, regenerative medicine options with injectable biologics have been trialed in discogenic pain and a wide variety of other painful musculoskeletal conditions. In particular, the role of bone marrow aspirate concentrate (BMAC) and culture-expanded bone marrow derived mesenchymal stromal cells (BM-MSCs) in treating discogenic pain remains unclear. The primary objective of this systematic review was to appraise the evidence of intradiscal injection with BMAC and culture-expanded BM-MSCs in alleviating pain intensity from discogenic pain. Secondary outcomes included changes in physical function after intradiscal injection, correlation between stromal cell count and pain intensity, and anatomical changes of the disc assessed by radiographic imaging after intradiscal injection. Overall, 16 studies consisting of 607 participants were included in qualitative synthesis without pooling. Our synthesis revealed that generally intradiscal autologous or allogeneic BMAC and culture-expanded BM-MSCs improved discogenic pain compared to baseline. Intradiscal injection was also associated with improvements in physical functioning and positive anatomical changes on spine magnetic resonance imaging (improved disc height, disc water content, Pfirrmann grading) although anatomical findings were inconsistent across studies. However, the overall GRADEscore for this study was very low due to heterogeneity and poor generalizability. There were no serious adverse events reported post intradiscal injection except for a case of discitis.

Multiplexed immunofluorescence identifies high stromal CD68+PD-L1+ macrophages as a predictor of improved survival in triple negative breast cancer

Triple negative breast cancer (TNBC) comprises 10–15% of all breast cancers and has a poor prognosis with a high risk of recurrence within 5 years. PD-L1 is an important biomarker for patient selection for immunotherapy but its cellular expression and co-localization within the tumour immune microenvironment and associated prognostic value is not well defined. We aimed to characterise the phenotypes of immune cells expressing PD-L1 and determine their association with overall survival (OS) and breast cancer-specific survival (BCSS). Using tissue microarrays from a retrospective cohort of TNBC patients from St George Hospital, Sydney (n = 244), multiplexed immunofluorescence (mIF) was used to assess staining for CD3, CD8, CD20, CD68, PD-1, PD-L1, FOXP3 and pan-cytokeratin on the Vectra Polaris™ platform and analysed using QuPath. Cox multivariate analyses showed high CD68+PD-L1+ stromal cell counts were associated with improved prognosis for OS (HR 0.56, 95% CI 0.33–0.95, p = 0.030) and BCSS (HR 0.47, 95% CI 0.25–0.88, p = 0.018) in the whole cohort and in patients receiving chemotherapy, improving incrementally upon the predictive value of PD-L1+ alone for BCSS. These data suggest that CD68+PD-L1+ status can provide clinically useful prognostic information to identify sub-groups of patients with good or poor prognosis and guide treatment decisions in TNBC.

Mesenchymal stromal cell count in bone marrow concentrate is not predicted by nucleated cell count or colony forming units

Purpose: Bone marrow aspirate concentrate (BMC) is a point-of-care tool for cartilage repair procedures. It is important to characterize biologics such as BMC both qualitatively and quantitatively so that a successful outcome can be attributed to a specific cellular or bioactive protein composition. Mesenchymal stromal cells (MSCs) are thought to contribute to the effectiveness of BMC, but they are somewhat difficult to quantify and are therefore not commonly characterized in clinical BMC samples. Nucleated cell count (NCC) and colony forming units (CFUs) have been proposed as surrogate markers to quantify MSCs in BMC. The purpose of this study was to test this assumption. We hypothesized that neither NCC nor CFUs would correlate with MSC quantity in BMC. Methods: Bone marrow was aspirated from the sternum of 15 horses (age 13.3±6 years; 4 castrated males, 11 females) and centrifuged to obtain BMC. Complete blood counts were obtained on each BMC sample. Two aliquots of BMC (0.5mL and 1mL) were plated in duplicate and cultured for up to 21 days when CFUs were counted. A CFU was defined as a colony measuring greater than or equal to1mm in diameter assessed by A) an ink stamp on inverted microscopy, or B) visual estimation after crystal violet staining. For each duplicate, one plate was stained with crystal violet, and cells on the other plate were lifted using Accumax (Innovative Cell Technologies, Inc, San Diego), counted, and viability was measured using trypan blue. Based on plastic adherence, these cells were presumed as MSCs and were subjected to confirmatory tri-lineage differentiation assays. A paired t-test was used to compare CFUs counted by inverted microscopy vs crystal violet methods. Bivariate correlation was used to investigate correlation between NCC & MSCs, NCC & CFUs, and CFU & MSCs. Statistical analysis was performed using JMP (SAS Institute, Cary, North Carolina, USA). Significance was set at P < 0.05. Results: Total NCC in BMC samples ranged from 2.5 - 320x103/μl, with a 128-fold variation in NCC, providing a wide range of data for analysis. CFUs were present on 59/68 plates; 9 plates showed no growth. There was no difference in CFUs counts between inverted microscopy and crystal violet methods (p=0.08). Presumptive MSCs ranged from 4 - 60x103 cells/plate. There was no or a very weak correlation between NCC and CFUs (Figure 1a; r=0.08, p=0.38), NCC and MSCs (Figure 1b; r=0.20, p=0.33), and CFUs and MSCs (Figure 1c; r=0.18, p=0.52). Cells were confirmed MSCs (n=10) based on ability to differentiate in adipogenic, osteogenic and chondrogenic lineages. Conclusions: Neither NCC nor CFU are appropriate surrogate methods for quantifying MSCs in BMC. There are several putative bioactive factors in BMC that could be responsible for its therapeutic effect, but to quantify MSC, laboratory-based methods such as tri-lineage differentiation or flow cytometry should be used to characterize BMC.

Does Mesenchymal Stromal Cell Count in Pre-autologous Hematopoietic Stem Cell Transplant Peripheral Blood and Apheresis Product Predict for Infectious Complications in the Post-transplant Period?

Mesenchymal stromal cells (MSC) have gained attention in the recent past considering their multipotentiality and organ-healing properties. Exogenous administration of MSC in the pre-hematopoietic stem cell transplant (HSCT) setting has been reported to enhance engraftment, heal graft-vs-host disease and increase infections in the post-HSCT period. In this study, we aimed to determine the effect of endogenous pre-HSCT MSC on the post-HSCT infectious complications in patients undergoing autologous-HSCT. The study included patients undergoing autologous-HSCT (n = 25; multiple myeloma-20, lymphoma-5). MSC were analyzed and quantified by flow cytometry in the peripheral blood (PB) at baseline, and in both PB and apheresis product (AP) following mobilization with growth factors. Pre-HSCT MSC (PB/AP) were correlated with the post-HSCT duration of febrile neutropenia and duration of antimicrobial drugs using Pearson's correlation co-efficient, and with the mucositis grade using Spearman's rank correlation. Pre-HSCT MSC (baseline and post-mobilization) correlated positively with the longer duration of febrile neutropenia and duration of antimicrobials used in the post-HSCT period (p < 0.05). Pre-HSCT MSC failed to correlate with post-HSCT engraftment and onset/severity/duration of oral and gastrointestinal mucositis. Endogenous pre-HSCT MSC counts might predict for increased infectious complications in the post autologous-HSCT setting.

Digital Image Analysis for Estimating Stromal CD8+ Tumor-Infiltrating Lymphocytes in Lung Adenocarcinoma

Background: Stromal CD8+ tumor-infiltrating lymphocytes (TILs) are an important prognostic and predictive indicator in non-small cell lung cancer (NSCLC). In this study, we aimed to develop and test the feasibility of a digital image analysis (DIA) workflow for estimating stromal CD8+ TIL density. Methods: A DIA workflow developed in a software platform (QuPath) was applied to a specified region of interest (ROI) within the stromal compartment of dual PD-L1/CD8 immunostained slides from 50 lung adenocarcinoma patients. A random tree classifier was trained from 25 training cases and applied to 25 test cases. The DIA-estimated CD8+ TIL densities were compared to manual estimates of three pathologists, who independently quantitated the percentage of CD8+ TILs from predefined ROIs in QuPath. Results: The average estimated total stromal cell count per case was 520 (range: 282–816) by QuPath and 551 (range: 265–744) by pathologists. The DIA-estimated CD8+ TIL density (mean = 16.9%) was comparable to pathologists’ manual estimates (mean = 15.9%). A paired t-test showed no statistically significant difference between DIA and pathologist estimates of CD8+ TIL density among both training (n = 25, P = 0.55) and test (n = 25, P = 0.34) cases. There was an almost perfect agreement between QuPath and each pathologist's estimates of CD8+ TIL density (κ = 0.85–0.86). Conclusions: These findings demonstrate the feasibility of applying a DIA workflow for estimating stromal CD8+ TIL density in NSCLC. DIA has the potential to provide an efficient and standardized approach for estimating stromal CD8+ TIL density.

Confocal biomicroscopic changes of the corneal layers following femtosecond laser-assisted MyoRing implantation in keratoconus

PurposeTo evaluate the effect of the femtosecond laser-assisted MyoRing implantation on the confocal biomicroscopic findings in different corneal layers of the patients with keratoconus. MethodsTwelve eyes of 12 patients with mild to moderate keratoconus (keratometry between 48 and 52 diopters) and intolerance to hard contact lens entered the study. All the included patients underwent femtosecond laser-assisted MyoRing (Dioptex GmBH, Linz, Austria) implantation. The confocal biomicroscopy of the cornea was performed for all corneal layers in the center and periphery preoperatively and 3 and 6 months postoperatively. The cell counts and the qualitative findings in each layer of the cornea were compared between preoperative and 3 and 6 months postoperative images. ResultsCompared with preoperative values, the central epithelial and the central and peripheral midstromal cell counts were significantly decreased 6 months after MyoRing implantation (P = 0.015, P = 0.010 and 0.005, respectively). Furthermore, compared with preoperative values, the peripheral posterior stromal cell count was significantly decreased 3 months after MyoRing implantation (P = 0.033). In the qualitative analysis, highly reflective nuclei in the basal epithelium, transient disruption in the subepithelial nerve plexus, increase in the reflectivity of the stromal keratocyte, and normal endothelial cell morphology were seen. ConclusionsOur study demonstrated some findings similar to that reported in intrastromal corneal ring segments (ICRS): decreased central epithelial cell counts, highly reflective nuclei in the basal epithelium, transient disruption in the subepithelial nerve plexus, and normal endothelial cell count and morphology. In addition, a decrease in the central and peripheral midstromal, transient decrease in posterior stromal cell counts, and absence of amorphous depositions were in contrast with the findings reported in ICRS.

Abstract P1-03-04: Response to cyclooxygenase-2 inhibition is regulated by collagen dense stroma

Abstract Women with dense breast have over four-fold risk for breast cancer and high breast density is correlated to increased collagen. In our previously characterized mouse model of MMTV-PyVT x Col1a1tm1jae, increased collagen levels in mammary tumors lead to enhanced tumor formation and progression. Additionally, high collagen density in vitro elevated expression of PTGS2, the gene for cyclooxygenase-2 (COX-2), by over four-fold. Because COX-2 over-expression is observed in 40% of invasive breast carcinoma cases and correlates with poor prognosis, we hypothesized that inhibition of COX-2 may be an effective target in the context of mammary tumors arising in dense tissue. Col1a1tm1jae (HD) or wild-type (wt) tumor mice were randomly assigned at 11 weeks of age to daily treatment with vehicle or celecoxib for 21 days. Tumors in HD mice were larger (p &amp;lt; 0.05) and expressed higher levels of COX-2 (p &amp;lt; 0.05) and PGE2 (p &amp;lt; 0.01). COX-2 and PGE2 expression levels were both decreased with COX-2 inhibition (wt and HD = p &amp;lt; 0.0001, for both markers). Cell proliferation decreased in both wt and HD tumors when COX-2 was inhibited (Epithelium wt and HD p &amp;lt; 0.0001; stroma wt = p &amp;lt; 0.05 and HD = p &amp;lt; 0.01). Notably, several cytokines, immune and stromal cell counts were elevated in HD tumors alone, and COX-2 inhibition reversed this effect. These findings indicate that COX-2 has a direct role in modulating tumor progression in dense matrices and it may be an effective therapeutic target for women with dense breast tissue. Citation Format: Esbona K, Inman DR, Saha S, Wilke LG, Keely PJ. Response to cyclooxygenase-2 inhibition is regulated by collagen dense stroma. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P1-03-04.

Effects of In-vivo Application of Cold Atmospheric Plasma on Corneal Wound Healing in New Zealand White Rabbits

Cold Atmospheric Plasma (CAP) has been shown to influence tissue wound healing but little is known about the impact of CAP on healthy corneal tissues and their ability to respond to injuries. The objective of this study is to examine the effect of CAP on wound healing after corneal epithelial and basement membrane ablation in New Zealand white rabbits. The rabbits were assigned into three groups. Ten Rabbits from two groups underwent a 6 mm corneal ablation to the right eyes. After ablation, five rabbits in group (A) received 2 minutes of CAP whereas the other five rabbits (B) were not treated with CAP. A third group (C) included two rabbits and received CAP without ablation. Eyes monitored for corneal haze, epithelial healing, lens clarity and any signs of inflammation. At 24 hours, two rabbits from group A and two from group B were sacrificed to harvest the corneas. Twenty days, all remaining rabbits in groups A, B, and C were sacrificed and corneas were harvested. Corneas were fixed in formalin and stained with H&E or used for immunofluorescence microscopy to assess scar formation using antibodies against fibronectin and a-smooth muscle actin. At 24 hours, corneas from group A had average epithelial defect of 9.25 mm2 on day 1 whereas those from group B had average defect of 12.05 mm2, a difference of 2.8 mm2 (P=0.57). H & E stained corneal sections didn’t show abnormal responses to injury at 24 hours and 20 days. Epithelial thickness and stromal cell counts 20 days after injury showed no significant differences. Analysis of immunofluorescence microscopy images showed no differences between all groups. In conclusion, CAP application to cornea doesn’t appear to have obvious adverse effects. CAP does not interfere with rate of wound closure or induce increased inflammation. CAP did not have an effect on corneal wound healing or lead to scar formation.

Endometrial cell counts in recurrent miscarriage: a comparison of counting methods

Studies of uterine natural killer (uNK) cells require reliable measurements of uNK cell density among diverse endometrial tissue. The aim of this study was to compare cell counting manually with two computer-aided methods based on a public domain software package, ImageJ. Immunohistochemistry (IHC) of CD56(+) uNK cells was performed on endometrium from recurrent miscarriage patients. Numbers of stromal cells per high-power field (HPF) were counted by two observers using: (i) manual tally counter and graticule; (ii) ImageJ 'point picker' tool; and (iii) ImageJ 'particle analysis' tool. Coefficients of variation (CV) and Bland-Altman plots were used to evaluate interobserver differences. Evaluation of %uNK using ImageJ particle analysis for stromal cell counts and point picker tool for uNK counts was undertaken. Point picker and particle analysis were significantly better than manual counting [interobserver CVs mean (standard deviation) 6.1% (3.3%); 4.7% (3.9%), 8.2% (6.5%), respectively]. Mean inter- and intra-observer CVs for %uNK were 10.3% (6.6%), 8.5% (4.9%) and 6.8% (4.3%), respectively. Bland-Altman analysis revealed no systematic differences in cell counts with the number of cells in the image for each method. Compared to manual cell counting, computer-aided image analysis yields more reproducible results for the assessment of uNK cells density using IHC.

High Harvest Yield, High Expansion, and Phenotype Stability of CD146 Mesenchymal Stromal Cells from Whole Primitive Human Umbilical Cord Tissue

Human umbilical cord blood is an excellent primitive source of noncontroversial stem cells for treatment of hematologic disorders; meanwhile, new stem cell candidates in the umbilical cord (UC) tissue could provide therapeutic cells for nonhematologic disorders. We show novel in situ characterization to identify and localize a panel of some markers expressed by mesenchymal stromal cells (MSCs; CD44, CD105, CD73, CD90) and CD146 in the UC. We describe enzymatic isolation and purification methods of different UC cell populations that do not require manual separation of the vessels and stroma of the coiled, helical-like UC tissue. Unique quantitation of in situ cell frequency and stromal cell counts upon harvest illustrate the potential to obtain high numerical yields with these methods. UC stromal cells can differentiate to the osteogenic and chondrogenic lineages and, under specific culturing conditions, they exhibit high expandability with unique long-term stability of their phenotype. The remarkable stability of the phenotype represents a novel finding for human MSCs, from any source, and supports the use of these cells as highly accessible stromal cells for both basic studies and potentially therapeutic applications such as allogeneic clinical use for musculoskeletal disorders.

Murine marrow stromal response to myelotoxic agents in vitro.

Previous studies have shown that the haemopoietically active murine long-term bone marrow culture (LTBMC) is a useful model for studying drug-induced suppression and recovery of myelopoiesis. We studied the effects on stromal morphology and stromal progenitors, assessed as colony forming unit-fibroblasts (CFU-F), of the addition of either the antimetabolite methotrexate (MTX) or the betalactam ceftazidime (CEF) to LTBMC. The examination of 500 micrograms/ml CEF-treated cultures revealed a stroma that appeared empty, with modest reduction in total stromal counts, and significant decreases in fat-containing and endothelial cells. In contrast, treatment with 1 microM MTX for 1 week initially caused minimal morphologic stromal changes, thereafter total stromal cell count as well as fibroblastoid, endothelial, fat containing and macrophage cells significantly increased. Haemopoiesis and the stroma recovered. Both CEF and MTX reversibly suppressed stromal progenitor cells in LTBMC. When added directly to CFU-F cultures, the concentrations resulting in a 50% colony growth inhibition were 214 micrograms/ml for CEF and 375 nM for MTX. These results suggest that CEF, but not MTX, has direct toxic effects on the stroma of established LTBMC. Stromal cell increase following MTX treatment probably indicates a stromal response that may contribute to haemopoietic cell recovery.

Melanocytes and iris color. Light microscopic findings.

To systematically evaluate morphologic differences in iris stroma that contribute to clinically perceptible differences in iris color, using immunohistochemical identification of stromal melanocytes and fluorescence microscopy. Paraffin-embedded sections from 51 human irides were stained with S100a and fluorescein isothiocyanate. Cells were counted and scored as melanocytes or other. Melanocyte number, proportion, and density were determined for light-colored (blue), medium-colored (hazel) and dark-colored (brown) irides and compared. No statistically significant difference was observed for mean total cellularity or mean melanocyte number among the three color groups. Mean total stromal cell count was 1177 +/- 259 (mean +/- SEM), and mean melanocyte number was 778 +/- 196 per 5-micrometer section. In human irides, 65.9% of the iris stroma is composed of melanocytes. Melanocyte density (number of cells per square millimeter) is not related to iris color. The number of melanocytes, the proportion of melanocytes, and iris stromal cellularity are not major contributors to iris color.

Postirradiation Hemopoietic Repopulation and Stromal Cell Viability

To evaluate some of the microenvironmental conditions necessary to support hemopoiesis, the right femurs of mice were x-irradiated. Femoral radioiron uptake in the right femur after 1000 rad fell to 20 +- 2 percent of the control (mean +- 1 SEM) at 3 days and returned to 67 +- 5 percent by 6 months. Erythroblast counts reflected these changes. Total nucleated cell counts initially diminished to 21 +- 3 percent of the control but increased to 76 +- 6 percent of normal by 6 months. Counts of bone marrow stromal cells in culture rose from 13 +- 4 percent of the control at 3 days to 77 +- 11 percent by 6 months. In contrast, except for temporary elevation in /sup 59/Fe uptake at 1 week, animals receiving 5000 or 10,000 rad to the right femur had persistently low levels (less than 30%) of total nucleated cell counts, stromal cell counts, /sup 59/Fe uptake, and erythroblast counts over 6 months. In the unirradiated marrow of these animals, the radioiron uptake and erythroblast counts were less than normal, and the total nucleated cell counts and splenic /sup 59/Fe uptake and weights were above normal through 6 months. We conclude that 5000more » or 10,000 rad inhibited sustained recovery of hemopoietic cells and stromal cells, 1000 rad permitted slightly subnormal hemopoietic repopulation and stromal cell recovery, the unirradiated marrow and spleen underwent dose- and time-dependent changes.« less