Cell-specific Markers Research Articles

Functional characterization of CD49f+ hepatic stem/progenitor cells in adult mice liver.

Hepatic Stem/progenitor cells (HSPCs) have gained a large amount of interest for treating acute liver disease. However, the isolation and identification of HSPCs are unclear due to the lack of cell-specific surface markers. To isolate adult HSPCs, we used cell surface-marking antibodies, including CD49f and Sca-1. Two subsets of putative HSPCs, Lin-CD45-Sca-1-CD49f+ (CD49f+) and Lin-CD45-Sca-1+CD49f- (Sca-1+) cells, were isolated from adult mice liver by flow cytometry. Robust proliferative activity and clonogenic activity were found in both CD49f+ and Sca-1+ cells through colony-forming tests and cell cycle analyses. Immunofluorescence staining revealed that CD49f+ cells expressed ALB and CK-19 while Sca-1+ cells expressed only ALB, indicating that CD49f+ cells were bipotential and capable of differentiating into hepatocyte and cholangiocyte. Consequently, PAS stain showed that differentiated CD49f+ and Sca-1+ cells synthesised glycogen, indicating they could differentiate into functional hepatocytes. mRNA expression profile indicated that both CD49f+ and Sca-1+ cells showed differential expression of genes that are associated with liver progenitor function such as Sox9 and EpCam. Moreover, two subsets of putative HSPCs were activated by DDC and we found that their abundance and proliferation increased with age. In summary, wehypothesized that CD49f+ cells were a type of potential HSPCs and may be utilised for clinical stem cell therapy.

A study of the immunohistochemical profile of bladder cancer in neuro-urological patients by the French Association of Urology.

To establish whether the expression of markers of cell differentiation (CK7, CK14, CK20, GATA3), apoptosis (p53), proliferation (Ki67, STAG2) and peri-tumoural lymphocytes (CD3, CD8), provides specific information about urothelial carcinogenesis in neuro-urological patients with bladder cancer (NBC). Tissue samples from NBC were retrieved from 15 centres in France and compared to control samples from non neuro-urological patients with bladder cancer (NNBC) and from neuro-urological patients without bladder cancer (NB). The expression of CK7, CK14, CK20, GATA3, p53, Ki67, STAG2, CD3 and CD8 markers was analysed using immunohistochemistry of tissue microarray sections. Overall, tissue samples from 124 patients were included in the study (n = 72 NBC, n = 26 NNBC and n = 26 NB). Muscle invasive bladder cancer (MIBC) was found in 52 NBC patients (72.2%) and squamous cell differentiation in 9 (12.5%). In NBC samples, the expression of CK20 and GATA3 was significantly more frequent in NMIBC compared to MIBC (p = 0.015 and p = 0.004, respectively). CK20 and GATA3 were significantly more expressed in NBC compared to NNBC (p < 0.001 and p = 0.010, respectively). The expression of CK14, Ki67, CD3 and CD8 was significantly more frequent in NBC than in NNBC samples (p = 0.005, p < 0.001, p < 0.001 and p < 0.001, respectively). The expression of CD3 and CD8 was similar in NBC and NB samples. In NBC, markers of basal differentiation, proliferation and peri-tumoural lymphocytes were significantly more expressed compared to NNBC controls. These results suggest the aggressiveness of NBC and the role of chronic inflammation in the carcinogenesis of bladder cancer in neuro-urological patients.

Peri-implant cell response on groove and pore-textured zirconia surfaces

ObjectivesThis study aimed to assess the independent influence of grooves and pores texturized by milling on gold-standard zirconia implant surfaces. MethodsMilled groove and pore textured with equivalent width, depth, and spacing on zirconia discs were produced using press and sintering techniques. All samples were sandblasted and acid-etched (SBAE), and untextured discs were used as controls. Osteoblasts and fibroblasts were cultured on discs for 14 days. Field emission gun-scanning electron microscopy (FEG-SEM) was used to observe cellular adhesion and morphology. Cell viability and proliferation assays were performed. Additionally, alkaline phosphatase activity, collagen type I, and osteopontin were evaluated at pre-defined time points. Results are presented as mean and standard deviation (SD), group comparisons were tested using one-way ANOVA (Tukey's post-hoc), and significance was set at P < 0.05. ResultsFEG-SEM images revealed cellular adhesion at 24 h in all samples with differences in distribution. Although both cell lines showed increased cell viability and differentiation cell markers such as collagen and osteopontin over time, statistically significant differences between groups were found in none of the quantitative study variables (P > 0.05). ConclusionThe results suggest similar cellular behavior between different patterns with similar dimensions and between them and microtopography by SBAE protocol currently used as the gold-standard for zirconia dental implants. The addition of pore and groove microtextures to the gold-standard zirconia dental implant surfaces treated with SBAE does not seem to be an asset in the cellular behavior of the hard and soft tissue cells.

Natural disaster and immunological aging in a nonhuman primate

Weather-related disasters are increasing in frequency and severity, leaving survivors to cope with ensuing mental, financial, and physical hardships. This adversity can exacerbate existing morbidities, trigger new ones, and increase the risk of mortality-features that are also characteristic of advanced age-inviting the hypothesis that extreme weather events may accelerate aging. To test this idea, we examined the impact of Hurricane Maria and its aftermath on immune cell gene expression in large, age-matched, cross-sectional samples from free-ranging rhesus macaques (Macaca mulatta) living on an isolated island. A cross section of macaques was sampled 1 to 4 y before (n = 435) and 1 y after (n = 108) the hurricane. Hurricane Maria was significantly associated with differential expression of 4% of immune-cell-expressed genes, and these effects were correlated with age-associated alterations in gene expression. We further found that individuals exposed to the hurricane had a gene expression profile that was, on average, 1.96 y older than individuals that were not-roughly equivalent to an increase in 7 to 8 y of a human life. Living through an intense hurricane and its aftermath was associated with expression of key immune genes, dysregulated proteostasis networks, and greater expression of inflammatory immune cell-specific marker genes. Together, our findings illuminate potential mechanisms through which the adversity unleashed by extreme weather and potentially other natural disasters might become biologically embedded, accelerate age-related molecular immune phenotypes, and ultimately contribute to earlier onset of disease and death.

Construction and Evaluation of Recombinant Chimeric Fibrillin and Elastin Fragment in Human Mesenchymal Stem Cells.

Diverse extracellular matrix (ECM) proteins physically interact with stem cells and regulate stem cell function. However, the large molecular weight of the natural ECM renders large-scale fabrication of a similar functional structure challenging. The objective of this study was to construct a low molecular weight and multifunctional chimeric form of recombinant ECM to stimulate mesenchymal stem cell (MSC) for tissue repair. We engineered Fibrillin-1PF14 fused to an elastin-like polypeptide to develop a new biomimetic ECM for stem cell differentiation and investigated whether this recombinant chimeric Fibrillin-Elastin fragment (rcFE) was effective on human nasal inferior turbinate-derived mesenchymal stem cells (hTMSCs). hTMSCs were grown in the medium supplemented with rcFE, then the effect of the protein was confirmed through cell adhesion assay, proliferation assay, and real-time PCR. rcFE enhanced the adhesion activity of hTMSCs by 2.7-fold at the optimal concentration, and the proliferation activity was 2.6-fold higher than that of the control group (non-treatment rcFE). In addition, when smooth muscle cell differentiation markers were identified by real-time PCR, Calponin increased about 6-fold, α-actin about 9-fold, and MYH11 about 10-fold compared to the control group. Chimeric rcFE enhanced cellular functions such as cell adhesion, proliferation, and smooth muscle differentiation of hTMSCs, suggesting that the rcFE can facilitate the induction of tissue regeneration.

Expression of Alpha Methyl Acyl-CoaRacemase (AMACR) Immunohistochemistry in Carcinoma Prostate in Tertiary Care Hospital

Objective: Prostate needle biopsy samples from cancer patients at a tertiary care hospital were analysed for the presence of the potential screening indicator alpha-methylacyl-COA RACEMES. Material and Methods: This was a Cross-sectional study carried out at Department of Histopathology, Lady Reading Hospital Peshawar from June, 2021 to December, 2021.Regardless of the patient's age, tumour grade, or histological type, all adenocarcinoma prostate specimens diagnosed at the Lady Reading Hospital Peshawar on the basis of immunohistochemistry and routine histology were examined. Standard and mean deviation were computed for quantitative factors of patient like age and frequencies, percentages were calculated for qualitative factors like AMACR expressions. Results: Outof 90 cases, AMACR positivity was found in 68 (85%), while negativity was seen in 22 (15%). Nine (11.3%) of the negative cases had one staining (weak, non-circumferential) and three cases (3%) had no stainings (No cytoplasmic staining). Conclusion: Positive AMACR staining on prostate needle core necropsies may be utilised to support a cancer diagnosis when the focus at issue is 1 mm in highest diameter. Basic hematoxylin and eosin standards for malice require analysis of cell-specific markers like P63 or 34E12. AMACR labelling must be interpreted in the context of other labels that can be used to find out more. Keywords: Adenocarcinoma of the prostate, Alphamethylacyl-CoA racemase (AMACR), Immunohistochemistry.

Effect of estrogen/progesterone ratio on the differentiation and the barrier function of epidermal keratinocyte and three-dimensional cultured human epidermis

AimsEstrogen (E) and progesterone (P) are the major female hormones and are secreted with changing concentration ratios throughout the menstrual cycle. These hormones have been studied individually regarding their physiological function in the skin, but their concentration ratio (E/P) and its effect on the skin has not yet been investigated. The purpose of this study was to clarify the effect of the E/P ratio on skin barrier function. The menstrual cycle was divided into the menstrual, follicular, ovulation, and luteal phases. Materials and methodsThe E/P concentration ratios corresponding with each phase were added to a three-dimensional epidermal model or normal human epidermal keratinocytes for 5 days. Gene and protein expression levels of several markers of cell differentiation, including loricrin (LOR) and transglutaminase (TGase), were quantified by real-time PCR and western blotting, respectively. Transepidermal water loss (TEWL) of the three-dimensional epidermal model was measured, and ceramide content was quantified by thin-layer chromatography. Key findingsGene expression of the epidermal differentiation markers, LOR and TGase, increased when applying the concentration ratio of E/P associated with the menstrual and luteal phases. The LOR protein level decreased from menstrual to luteal phases, and the TGase protein level increased from menstrual to luteal phases. During the same phases, ceramide NS increased and TEWL decreased. SignificanceSkin barrier function was improved by culturing cells at specific E/P concentration ratios, which would, therefore, be considered beneficial for female skin. This suggests that dysregulated E/P concentration ratios may be the cause of certain skin problems.

METHYLTRANSFERASE SMYD2 MODULATES THERAPEUTIC EFFECTS OF EPIDERMAL GROWTH FACTOR IN INFLAMMATORY BOWEL DISEASE

Abstract BACKGROUND Inflammatory bowel disease (IBD) is a chronic and remitting inflammatory disorder of the gastrointestinal tract. A prior clinical trial demonstrated the ability of epidermal growth factor receptor (EGFR) ligand EGF to induce and maintain remission in ulcerative colitis. As a protein lysine methyltransferase, SET and MYND domain-containing protein 2 (SMYD2) regulates various cellular functions, including chromatin remodeling and tumorigenesis. A bioinformatic study has shown that EGFR might be a candidate for SMYD2-mediated methylation. In this research, we aimed to investigate whether SMYD2 depletion could synergize with EGFR-activating treatment to promote intestinal goblet cell regeneration in IBD. METHODS SMYD2 flox C57BL/6 (SMYD2fl/fl) and intestine epithelial cell-specific conditional knockout of SMYD2 (SMYD2ΔIEC) male mice (8 to10-week old) were given 3 cycles of DSS treatment, each cycle including 5 days of 2% DSS in drinking water followed by 16 days of normal drinking water. EGF (50 μg/kg/day) was administered via intraperitoneal injection once per day for 6 days during the third cycle of DSS treatment (from day 42 to 47 along with DSS administration). The disease activity index (DAI), including body weight loss, stool consistency and colorectal bleeding, was monitored and calculated daily. Histologic scoring was used to evaluate the extent of histopathologic change in middle colon tissue. Goblet cell differentiation and maturation was analyzed via Alcian Blue/periodic acid-Schiff (AB/PAS) staining and immunostaining. RESULTS EGF treatment significantly increased the number of goblet cells (AB/PAS staining positive cells) in both SMYD2fl/fl and SMYD2ΔIEC mice. However, in SMYD2ΔIEC mice, more mature goblet cells were observed. In contrast, in SMYD2fl/fl mice, most goblet cells were accumulated at the lower half of colon crypts with small mucin particles. As compared with SMYD2fl/fl mice receiving EGF treatment, SMYD2 depletion combined with EGF injection (6 days) failed to significantly improve body weight loss and DAI scoring during DSS treatment, as well as histologic scoring (including inflammatory cell infiltration and epithelial tissue damage). Immunostaining of goblet cell differentiation markers (including ATOH1, SPDEF and KLF4) revealed no significant difference between SMYD2fl/fl and SMYD2ΔIEC mice. CONCLUSION SMYD2 depletion combined with EGF treatment could promote goblet cell expansion and maturation in mouse DSS-induced chronic colitis. However, the mechanism by which SMYD2 depletion facilitates goblet cell maturation needs further investigation.

Sodium/hydrogen-exchanger-2 modulates colonocyte lineage differentiation.

The sodium/hydrogen exchanger 2 (NHE2) is an intestinal acid extruder with crypt-predominant localization and unresolved physiological significance. Our aim was to decipher its role in colonic epithelial cell proliferation, differentiation and electrolyte transport. Alterations induced by NHE2-deficiency were addressed in murine nhe2-/- and nhe2+/+ colonic crypts and colonoids, and NHE2-knockdown and control Caco2Bbe cells using pH-fluorometry, gene expression analysis and immunofluorescence. pHi -measurements along the colonic cryptal axis revealed significantly decreased intracellular pH (pHi ) in the middle segment of nhe2-/- compared to nhe2+/+ crypts. Increased Nhe2 mRNA expression was detected in murine colonoids in the transiently amplifying/progenitor cell stage (TA/PE). Lack of Nhe2 altered the differentiation programme of colonic epithelial cells with reduced expression of absorptive lineage markers alkaline phosphatase (iAlp), Slc26a3 and transcription factor hairy and enhancer-of-split 1 (Hes1), but increased expression of secretory lineage markers Mucin 2, trefoil factor 3 (Tff3), enteroendocrine marker chromogranin A and murine atonal homolog 1 (Math1). Enterocyte differentiation was found to be pHi dependent with acidic pHi reducing, and alkaline pHi stimulating the expression of enterocyte differentiation markers in Caco2Bbe cells. A thicker mucus layer, longer crypts and an expanded brush border membrane zone of sodium/hydrogen exchanger 3 (NHE3) abundance may explain the lack of inflammation and the normal fluid absorptive rate in nhe2-/- colon. The results suggest that NHE2 expression is activated when colonocytes emerge from the stem cell niche. Its activity increases progenitor cell pHi and thereby supports absorptive enterocyte differentiation.

Changes in Expression of Specific mRNA Transcripts after Single- or Re-Irradiation in Mouse Testes

Alkylating agents and irradiation induce testicular damage, which results in prolonged azoospermia. Even very low doses of radiation can significantly impair testis function. However, re-irradiation is an effective strategy for locally targeted treatments and the pain response and has seen important advances in the field of radiation oncology. At present, little is known about the relationship between the harmful effects and accumulated dose of irradiation derived from continuous low-dose radiation exposure. In this study, we examined the levels of mRNA transcripts encoding markers of 13 markers of germ cell differentiation and 28 Sertoli cell-specific products in single- and re-irradiated mice. Our results demonstrated that re-irradiation induced significantly decreased testicular weights with a significant decrease in germ cell differentiation mRNA species (Spo11, Tnp1, Gfra1, Oct4, Sycp3, Ddx4, Boll, Crem, Prm1, and Acrosin). In the 13 Sertoli cell-specific mRNA species decreased upon irradiation, six mRNA species (Claudin-11, Espn, Fshr, GATA1, Inhbb, and Wt1) showed significant differences between single- and re-irradiation. At the same time, different decreases in Sertoli cell-specific mRNA species were found in single-irradiation (Aqp8, Clu, Cst12, and Wnt5a) and re-irradiation (Tjp1, occludin, ZO-1, and ZO-2) mice. These results indicate that long-term aspermatogenesis may differ after single- and re-irradiated treatment.

Postmortem high-dimensional immune profiling of severe COVID-19 patients reveals distinct patterns of immunosuppression and immunoactivation

A complete diagnostic autopsy is the gold-standard to gain insight into Coronavirus disease 2019 (COVID-19) pathogenesis. To delineate the in situ immune responses to SARS-CoV-2 viral infection, here we perform comprehensive high-dimensional transcriptional and spatial immune profiling in 22 COVID-19 decedents from Wuhan, China. We find TIM-3-mediated and PD-1-mediated immunosuppression as a hallmark of severe COVID-19, particularly in men, with PD-1+ cells being proximal rather than distal to TIM-3+ cells. Concurrently, lymphocytes are distal, while activated myeloid cells are proximal, to SARS-CoV-2 viral antigens, consistent with prevalent SARS-CoV-2 infection of myeloid cells in multiple organs. Finally, viral load positively correlates with specific immunosuppression and dendritic cell markers. In summary, our data show that SARS-CoV-2 viral infection induces lymphocyte suppression yet myeloid activation in severe COVID-19, so these two cell types likely have distinct functions in severe COVID-19 disease progression, and should be targeted differently for therapy.

In vitro Culture and Morphometry of Porcine Adipose Derived Mesenchymal Stem Cells (pAD-MSCs)

Background: Mesenchymal stem cells are well known for their self-renewal capacity and ability to differentiate into multiple cell lineages. The aim of the study was to develop a simple technique for isolation of mesenchymal stem cells from porcine adipose tissue and to study the morphometric characteristics of porcine mesenchymal stem cells. Methods: Porcine adipose derived mesenchymal stem cells were isolated in vitro by using collagenase type II enzyme. Cell yield and viability of the cells were calculated by using trypan blue exclusion method using Neubauer’s chamber. Characterization of MSCs were done by using specific cell markers. The morphological changes, morphometry were analysed in culture using Leishman’s stain. The cell doubling (CD) and Population doubling time (PDT) were also calculated. Result: The isolated adherent cells start forming colony and demonstrated an elongated, round and spindle like fibroblastic morphology by day 1. Almost 80-90 per cent confluency was attained on day 8-9 after the initial seeding and was reduced to day 3-4 in the subsequent passages. RT-PCR reactions revealed positive expression of mesenchymal stem cell markers CD44, CD73 and negative expression of CD34, a hematopoietic cell surface marker. Immunocytochemistry also revealed positive expression for CD44 and negative for CD34. In morphometric studies, the cell length, nucleus length, cell width and nucleus width were increased between 24 and 48 hours in both P2 and P3.

Combined overexpression of four transcription factors promotes effector T cell dedifferentiation toward early phenotypes.

Effector T cells, which are abundant but are short-lived after reinfusion into the body, are generally used for T-cell therapy, and antitumor immunity is typically not maintained over the long term. Genetic modification by early differentiated T cells and reinfusion has been shown to enhance antitumor immunity in vivo. This study overexpressed the characteristic transcription factors of differentiated early T cells by transfecting effector T cells with transcription factor recombinant lentivirus (S6 group: BCL6, EOMES, FOXP1, LEF1, TCF7, KLF7; S1 group: BCL6, EOMES, FOXP1, KLF7; S3 group: BCL6, EOMES, FOXP1, LEF1) to induce a sufficient number of effector T cells to dedifferentiate and optimize the transcription factor system. The results revealed that overexpression of early characteristic transcription factors in effector T cells upregulated the expression of early T cell differentiation markers (CCR7 and CD62L), with the S1 group having the highest expression level, while the rising trend of late differentiation marker (CD45RO) expression was suppressed. Moreover, the expression of early differentiation-related genes (ACTN1, CERS6, BCL2) was significantly increased, while the expression of late differentiation-related genes (KLRG-1) and effector function-related genes (GNLY, GZMB, PRF1) was significantly decreased; this difference in expression was more significant in the S1 group than in the other two experimental groups. The antiapoptotic ability of each experimental group was significantly enhanced, while the secretion ability of TNF-α and IFN-γ was weakened, with the effector cytokine secretion ability of the S1 group being the weakest. Transcriptomic analysis showed that the gene expression profile of each experimental group was significantly different from that of the control group, with differences in the gene expression pattern and number of differentially expressed genes in the S1 group compared with the other two experimental groups. The differentially expressed gene enrichment pathways were basically related to the cell cycle, cell division, and immune function. In conclusion, overexpression of early characteristic transcription factors in effector T cells induces their dedifferentiation, and induction of dedifferentiation by the S1 group may be more effective.

CRISPR/Cas9 editing of directly reprogrammed myogenic progenitors restores dystrophin expression in a mouse model of muscular dystrophy.

SummaryGenetic mutations in dystrophin manifest in Duchenne muscular dystrophy (DMD), the most commonly inherited muscle disease. Here, we report on reprogramming of fibroblasts from two DMD mouse models into induced myogenic progenitor cells (iMPCs) by MyoD overexpression in concert with small molecule treatment. DMD iMPCs proliferate extensively, while expressing myogenic stem cell markers including Pax7 and Myf5. Additionally, DMD iMPCs readily give rise to multinucleated myofibers that express mature skeletal muscle markers; however, they lack DYSTROPHIN expression. Utilizing an exon skipping-based approach with CRISPR/Cas9, we report on genetic correction of the dystrophin mutation in DMD iMPCs and restoration of protein expression in vitro. Furthermore, engraftment of corrected DMD iMPCs into the muscles of dystrophic mice restored DYSTROPHIN expression and contributed to the muscle stem cell reservoir. Collectively, our findings report on a novel in vitro cellular model for DMD and utilize it in conjunction with gene editing to restore DYSTROPHIN expression in vivo.

MiR-27a-3p Targets GLP1R to Regulate Differentiation, Autophagy, and Release of Inflammatory Factors in Pre-Osteoblasts via the AMPK Signaling Pathway.

Objective: Osteoporosis is caused by the dysregulation of bone homeostasis which is synergistically mediated by osteoclasts and osteoblasts. MiR-27a-3p is a key inhibitor of bone formation. Hence, unearthing the downstream target gene of miR-27a-3p is of great significance to understand the molecular mechanism of osteoporosis. Methods: Bioinformatics analysis was utilized to find the downstream target gene of miR-27a-3p, and dual-luciferase reporter assay was conducted to validate the interplay of miR-27a-3p and GLP1R. Besides, qRT-PCR, Western blot, and enzyme-linked immunosorbent assay (ELISA) were employed to verify the impact of miR-27a-3p on GLP1R expression and the differentiation, autophagy, and inflammatory response of MC3T3-E1 pre-osteoblasts. Results: Dual-luciferase assay validated that miR-27a-3p directly targeted GLP1R. Additionally, posttreatment of MC3T3-E1 cells with miR-27a-3p mimics resulted in a remarkable decrease in expression levels of GLP1R, cell differentiation marker gene, autophagy marker gene, and AMPK. These results indicated that miR-27a-3p targeted GLP1R to inhibit AMPK signal activation and pre-osteoblast differentiation and autophagy, while promoting the release of inflammatory factors. Conclusion: The miR-27a-3p/GLP1R regulatory axis in pre-osteoblasts contributes to understanding the molecular mechanism of osteoporosis.

Abstract P077: Composition of CD4 T cell subsets and impact on tumor growth control across mouse syngeneic tumor models

Abstract CD4 T cells include multiple sublineages orchestrating a broad range of effector activities during the initiation, promotion, and progression of carcinogenesis. Although regulatory T cells (Tregs) are well-characterized to promote tumor progression, the impact of effector CD4+ T cell subsets (Teff) in anti-tumor immunity is less well known. Further, the relative contribution of Teff and Treg cell subsets in different syngeneic tumor models has not been characterized. We observed that CD4 depletion had significantly different impact of tumor growth across different syngeneic tumor models: no effect on MC38 tumor growth; enhanced tumor growth in CT26.KSA tumor model; while almost complete tumor regression in the EMT-6 tumor model. We hypothesized that each tumor model has a vastly different composition in the CD4 T cell compartment. To test this, we characterized expression of activation and CD4 T cell differentiation markers as well as cytokine production from CD4 TILs in each of the syngeneic tumor models. We found that while effector CD4 TILs (FoxP3−CD4+) from MC38 and CT26.KSA tumors expressed high levels activation and differentiation markers, Teff from EMT-6 tumors expressed very low levels of T cell activation and differentiation markers, suggesting the Teff in EMT-6 tumors are largely dysfunctional. Ex vivo restimulation of tumor samples with anti-CD3/anti-CD28 promoted IFNγ production in CD4 TILs from MC38 and CT26.KSA tumor models, but not from EMT-6 tumor models, further supporting that Teff from MC38 and CT26.KSA tumors remain highly active, while CD4 TILs from EMT-6 tumors are dysfunctional. When looking at the Treg compartment, the frequency of Tregs in CT26.KSA tumors was reduced compared to MC38 and EMT-6 tumors. Further, Tregs in MC38 and EMT-6 tumors expressed very high levels of CD69, a marker of highly immunosuppressive Tregs, while Tregs in CT26.KSA tumors expressed lower levels of CD69, suggesting that these Tregs may be less immunosuppressive. In summary, the CD4 T cell compartment is vastly different across CT26.KSA, MC38 and EMT-6 syngeneic tumor models, resulting in very different impact of tumor growth control. Citation Format: Chunxiao Xu, Lindsay Webb, Sireesha Yalavarthi, Clotilde Bourin, Jacques Moisan. Composition of CD4 T cell subsets and impact on tumor growth control across mouse syngeneic tumor models [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P077.

Uncertain histological origin of "glia-like" cells in adult human brain tissue cultures.

Most brain cells studies come from cultured rodent brain tissue, so basic questions about the behaviour of cultured adult human glial cells may remain unanswered. Cells cultured from adult human brain have been poorly defined until now and are often termed "glia-like" based on some morphological similarities with astrocytes. However, the cells in question fail to express glial markers and may be well be of non-glial origin. We examined adult human brain and cultures from 10 patients with non-malignant diagnoses. Immunofluorescence methods were used for glial and non-glial cell type identifications. Confluent cultures contained the following: 0.1 % astrocytes, ≤ 0.01 % oligodendrocytes, 2-5 % microglial and 95-98 % "glia-like" cells. Astrocytes tested as followed: GFAP+/Vim+, microglia: Ferr+Vim+, "glia-like" cells: Vim+/Fn+/CK- or CK+. In the brain tissue, astrocytes were GFAP+/Vim+, microglia Ferr+/Vim-, fibronectin expression was restricted to brain vessels. This report demonstrates considerable morphological and cytoskelatal dedifferentiation of cultured brain cells. Cytokeratins, specific markers for epithelial cell differentiation, were absent in the brain tissue. However, they were expressed in "glia-like" cells. This finding could be considered glial dedifferentiation given the ectodermal origin of the brain tissue. We suggest that "glia-like" cells come from currently unknown glial progenitor cells scattered through the brain tissue (Tab. 1, Fig. 4, Ref. 19).

Loss of function of GATA3 induces basal-like mammary tumors.

Purpose: GATA3 is a transcription factor essential for mammary luminal epithelial cell differentiation. Expression of GATA3 is absent or significantly reduced in basal-like breast cancers. Gata3 loss-of-function impairs cell proliferation, making it difficult to investigate the role of GATA3 deficiency in vivo. We previously demonstrated that CDK inhibitor p18INK4c (p18) is a downstream target of GATA3 and restrains mammary epithelial cell proliferation and tumorigenesis. Whether and how loss-of-function of GATA3 results in basal-like breast cancers remains elusive.Methods: We generated mutant mouse strains with heterozygous germline deletion of Gata3 in p18 deficient backgrounds and developed a Gata3 depleted mammary tumor model system to determine the role of Gata3 loss in controlling cell proliferation and aberrant differentiation in mammary tumor development and progression.Results: Haploid loss of Gata3 reduced mammary epithelial cell proliferation with induction of p18, impaired luminal differentiation, and promoted basal differentiation in mammary glands. p18 deficiency induced luminal type mammary tumors and rescued the proliferative defect caused by haploid loss of Gata3. Haploid loss of Gata3 accelerated p18 deficient mammary tumor development and changed the properties of these tumors, resulting in their malignant and luminal-to-basal transformation. Expression of Gata3 negatively correlated with basal differentiation markers in MMTV-PyMT mammary tumor cells. Depletion of Gata3 in luminal tumor cells also reduced cell proliferation with induction of p18 and promoted basal differentiation. We confirmed that expression of GATA3 and basal markers are inversely correlated in human basal-like breast cancers.Conclusions: This study provides the first genetic evidence demonstrating that loss-of-function of GATA3 directly induces basal-like breast cancer. Our finding suggests that basal-like breast cancer may also originate from luminal type cancer.

Abstract PO-157: Calcium/calmodulin-dependent protein kinase-related peptide (CARP) increases stemness of colorectal cancer cells

Abstract Calcium/calmodulin-dependent protein kinase-related peptide (CARP) is one of the five alternative splicing products of the doublecortin-like kinase 1 (DCLK1) gene, which is a specific marker for cancer stem cells in multiple cancers. Up-regulation of DCLK1 is correlated with progression and poor prognosis of malignant cancers, but the role of individual DCLK1 isoforms during tumorigenesis is unclear. CARP was reported to play an important role in the hippocampus and in the facilitation of apoptosis in granule cells of the rat dentate gyrus. Here we aimed to reveal the effects of CARP on the stemness of cancer cells. In order to achieve our goal, isogenic CARP over-expressing cells were established using HCT116 cells, a colorectal cancer cell line. Spheroid formation assay, proliferation assay, and clonogenic capacity assay were applied to assess the effects of CARP on cancer biology. Our results demonstrated that over-expression of CARP increased the number of spheroids, but decreased the proliferation rate in comparison to parental control HCT116 cells. In conclusion, CARP can increase the self-renewal capability of cancer cells under spheroid formation conditions, but can also inhibit cell proliferation under normal cell culture conditions. CARP may be used to develop a therapeutic target for cancer treatment. Citation Format: Leo Mei, Jinghe Mao. Calcium/calmodulin-dependent protein kinase-related peptide (CARP) increases stemness of colorectal cancer cells [abstract]. In: Proceedings of the AACR Virtual Conference: 14th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2021 Oct 6-8. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr PO-157.

Isolation of tdTomato Expressing Inter-follicular Epidermal Melanocytes or Keratinocytes from Mouse Tail Skin.

The epidermis is the outermost layer of the skin. It is made up of mostly keratinocytes along with a small number of melanocytes and Langerhans cells. Melanocytes produce a pigment called melanin, which is transferred to the keratinocytes, and protects these cells from damage from UV radiation, as well as generating hair and skin colours. In this important relationship, keratinocytes exert control over melanocytes. Many questions regarding keratinocyte-melanocyte interactions have yet to be answered, and would benefit from study in model systems, to address diseases such as vitiligo and cutaneous melanoma. Most of the mouse is covered in fur and these areas lack the skin pigmenting inter-follicular epidermal (IFE) melanocytes. However, the mouse tail is pigmented analogously to human skin. Here, we present a method for isolating IFE melanocytes or keratinocytes expressing the tdTomato marker from the mouse tail, using fluorescence-activated cell sorting (FACS). The method involves firstly separating the tail skin epidermis from the dermis, and then digesting the epidermis to produce dissociated cells, which can then be sorted. These isolated cell populations can be studied using RNAseq or cultured in vitro. This protocol isolates IFE melanocytes or keratinocytes and immediately provides reasonable yields of cells, without the need to stain the cells for cell specific markers.