Cell Markers CD133 Research Articles

Machine learning methods for diagnostic disease prediction and treatment decisions in parvovirus B19 positive viral cardiomyopathy

Abstract Background Parvovirus B19 (B19V) is the most common type of virus found in endomyocardial biopsies (EMB) from patients with inflammatory heart disease. However, the detection of genomic B19V-DNA appears to have little clinical significance and is often considered an incidental finding. Recent analyses of a large cohort of B19V-positive patients revealed that the detection of RNA transcripts rather than DNA is of clinical importance, as B19V-RNA-positive patients have a significantly worse cardiovascular outcome. Here, we applied machine learning (ML) methods to assess the impact of B19V transcriptional activity on cardiac inflammation and patient clinical outcome. Methods and results A total of 305 patients with a known clinical course and a positive endomyocardial B19V-DNA test were included in this study. Follow-up measurements of the B19V-specific transcripts VP1/2 and NS1 revealed significantly worse clinical courses than in patients without B19V-RNA detection (P=0.019, hazard ratio (95% CI)=1.62 (1.10-2.57)). LVEF at follow-up was significantly lower in patients with detected B19V-RNA (P=0.007), and adverse events such as worsening LV function (P=0.003) or stable systolic dysfunction (P=0.013) were more frequent in this group. Importantly, analysis of inflammatory markers in EMB showed no difference in patients with and without RNA detection (P=0.432), suggesting that the deterioration in clinical outcome was related to B19V transcriptional activity rather than an increase in inflammation. The analysis of area under the receiver operating characteristic curve (AUROC) of B19V-RNA-negative patients revealed that the macrophage marker MAC-1 (AUROC=0.66, P=0.002) was the single most prognostically relevant parameter, while the T cell marker CD3 (AUROC=0.73, P<0.001) was identified as the most prognostically relevant marker in B19V-RNA-positive patients. The linear combination of several inflammatory markers increased the AUROC value to 0.67 in B19V-RNA-negative patients and to 0.74 in B19V-RNA-positive patients. To further increase prognostic accuracy, we trained ML models on features derived from EMB inflammatory markers. For the prediction of major adverse cardiac events within 60 months, the best ML-models achieved an AUROC of 0.87 in B19V-RNA-positive and of 0.86 in B19V-RNA-negative patients, while models that neglect the differentiation of patients with regard to the presence of B19V RNA transcripts only achieved an AUROC of up to 0.76. Conclusions These results show that B19V transcriptional activity is an independent risk marker for adverse cardiac events in patients with viral cardiomyopathy and thus confirm the clinical-therapeutic importance of myocardial biopsy. Furthermore, we present an ML-based algorithm that accurately indicates disease progression in B19V-positive cardiomyopathy patients and is thus helpful for appropriate therapy recommendation.

Abstract We053: Altered inflammatory state and mitochondrial function identified by transcriptomics in paediatric congenital heart patients prior to surgical repair

Background: Congenital heart disease (CHD) remains the most common birth defect, with surgical intervention required in complex cases. In contrast to adult heart disease which disproportionately affects the left ventricle, complex CHD can be characterized by increased right ventricular (RV) pressure, leading to RV hypertrophy and eventually failure, with RV function known to be a major predictor in sustained cardiac health in these patients. Methods: RV samples were collected from discarded myocardial tissue from paediatric patients (0-16 years) undergoing their first cardiac surgery at Bristol Royal Hospital for Children or obtained from autopsy subjects. RNA sequencing and subsequent cellular deconvolution was conducted in CHD samples and integrated with publicly available control dataset. Cellular deconvolution, pathway network analysis and gene ontology were conducted and changes in expression of markers was confirmed via histological analysis. Results: Presence of CHD drove divergence of transcriptome with 511 differentially expressed genes (log fold change≥±2, padj≤0.05), with 82% shared homology. Known CHD genes; TBX5 , JAG1, NOTCH2 and NOTCH1 were reduced in CHD compared to controls (padj=2.24 E-07 , 1.38 E-27 , 7.76 E-31 , 9.11 E-7 ) . Conversely, mitochondrial dysfunction genes RPPH1 and RMPR (padj=4.67 E-132 , 2.23 E-107 ) were increased. Gene set enrichment identified mitochondrial dysfunctional pathways.Negative regulation of mitochondrial functions and metabolism was identified in functional network analysis. T helper cell markers CD4 and SELL were significantly downregulated (p<0.0001, p=0.003). Cytotoxic T cell markers CD8a, LAGE3 and CD49a were significantly increased in CHD compared to control (p=0.0006, p<0.0001, p=0.0118) as was proinflammatory marker Caspase1 (p=0.0055). Histological analysis confirmed an increase in CD45 and CD8 positive cellular bodies with the CHD tissue, which was absent in control. Deconvolution of bulk RNAseq data suggests a reduction in CD4+ T cells (p=0.0067) and an increase in CD8+ T cells (p=0.0223). Network analysis identified positive regulation of the immune system and cytokine signalling clusters within the inflammation functional network as were lymphocyte activation and leukocyte differentiation. Conclusions: Utilizing RV tissue from paediatric patients undergoing CHD cardiac surgery this study identifies dysfunctional mitochondrial pathways and an increase in inflammatory T cell presence prior to reparative surgery.

Culture and Immunomodulation of Equine Muscle-Derived Mesenchymal Stromal Cells: A Comparative Study of Innovative 2D versus 3D Models Using Equine Platelet Lysate.

Muscle-derived mesenchymal stromal cells (mdMSCs) hold great promise in regenerative medicine due to their immunomodulatory properties, multipotent differentiation capacity and ease of collection. However, traditional in vitro expansion methods use fetal bovine serum (FBS) and have numerous limitations including ethical concerns, batch-to-batch variability, immunogenicity, xenogenic contamination and regulatory compliance issues. This study investigates the use of 10% equine platelet lysate (ePL) obtained by plasmapheresis as a substitute for FBS in the culture of mdMSCs in innovative 2D and 3D models. Using muscle microbiopsies as the primary cell source in both models showed promising results. Initial investigations indicated that small variations in heparin concentration in 2D cultures strongly influenced medium coagulation with an optimal proliferation observed at final heparin concentrations of 1.44 IU/mL. The two novel models investigated showed that expansion of mdMSCs is achievable. At the end of expansion, the 3D model revealed a higher total number of cells harvested (64.60 ± 5.32 million) compared to the 2D culture (57.20 ± 7.66 million). Trilineage differentiation assays confirmed the multipotency (osteoblasts, chondroblasts and adipocytes) of the mdMSCs generated in both models with no significant difference observed. Immunophenotyping confirmed the expression of the mesenchymal stem cell (MSC) markers CD-90 and CD-44, with low expression of CD-45 and MHCII markers for mdMSCs derived from the two models. The generated mdMSCs also had great immunomodulatory properties. Specific immunological extraction followed by enzymatic detection (SIEFED) analysis demonstrated that mdMSCs from both models inhibited myeloperoxidase (MPO) activity in a strong dose-dependent manner. Moreover, they were also able to reduce reactive oxygen species (ROS) activity, with mdMSCs from the 3D model showing significantly higher dose-dependent inhibition compared to the 2D model. These results highlighted for the first time the feasibility and efficacy of using 10% ePL for mdMSC expansion in novel 2D and 3D approaches and also that mdMSCs have strong immunomodulatory properties that can be exploited to advance the field of regenerative medicine and cell therapy instead of using FBS with all its drawbacks.

Carotid artery transplantation of brain endothelial cells enhances neuroprotection and neurorepair in ischaemic stroke rats.

Brain microvascular endothelial cells (BMECs), an important component of the neurovascular unit, can promote angiogenesis and synaptic formation in ischaemic mice after brain parenchyma transplantation. Since the therapeutic efficacy of cell-based therapies depends on the extent of transplanted cell residence in the target tissue and cell migration ability, the delivery route has become a hot research topic. In this study, we investigated the effects of carotid artery transplantation of BMECs on neuronal injury, neurorepair, and neurological dysfunction in rats after cerebral ischaemic attack. Purified passage 1 endothelial cells (P1-BMECs) were prepared from mouse brain tissue. Adult rats were subjected to transient middle cerebral artery occlusion (MCAO) for 30 min. Then, the rats were treated with 5 × 105 P1-BMECs through carotid artery infusion or tail vein injection. We observed that carotid artery transplantation of BMECs produced more potent neuroprotective effects than caudal injection in MCAO rats, including reducing infarct size and alleviating neurological deficits in behavioural tests. Carotid artery-transplanted BMECs displayed a wider distribution in the ischaemic rat brain. Immunostaining for endothelial progenitor cells and the mature endothelial cell markers CD34 and RECA-1 showed that carotid artery transplantation of BMECs significantly increased angiogenesis. Carotid artery transplantation of BMECs significantly increased the number of surviving neurons, decreased the cerebral infarction volume, and alleviated neurological deficits. In addition, we found that carotid artery transplantation of BMECs significantly enhanced ischaemia-induced hippocampal neurogenesis, as measured by doublecortin (DCX) and Ki67 double staining within 2 weeks after ischaemic injury. We conclude that carotid artery transplantation of BMECs can promote cerebral angiogenesis, neurogenesis, and neurological function recovery in adult rats after ischaemic stroke. Our results suggest that carotid injection of BMECs may be a promising new approach for treating acute brain injuries.

P-736 The therapeutic effect of hysteroscopic surgery on chronic endometritis in infertile women with intrauterine disorders

Abstract Study question Can chronic endometritis (CE) be treated by hysteroscopic surgery without antibiotic treatment in women with intrauterine disorders? Summary answer Most CE cases with intrauterine disorders were cured through hysteroscopic surgery without antibiotic therapy, regardless of the type of intrauterine abnormalities. What is known already Persistent inflammation of the local endometrium, CE, interferes with embryo implantation. CE is primarily caused by intrauterine infection; therefore, broad-spectrum antibiotic therapy is the current treatment for CE. Nevertheless, most cases with CE and endometrial polyps can be cured via hysteroscopic polypectomy without antibiotic therapy in our previous study. Study design, size, duration This study was a prospective cohort study approved by the Ethics Committee. To evaluate the therapeutic effect of hysteroscopic surgery on CE, the endometrial specimens for the immunohistochemistry analysis of the plasma cell marker CD138 were obtained to diagnose CE at surgery between November 2018 and June 2021. In the women diagnosed with CE, endometrial biopsy was performed without use of antibiotics in the subsequent menstrual cycle. Participants/materials, setting, methods The study population consisted of 337 consecutive infertile women who underwent hysteroscopic surgery. Eighty-nine consecutive patients without intrauterine disorders were also recruited as control group. CE was diagnosed as the presence of ≥ 5 CD138-positive cells in 10 nonoverlapping random stromal areas of high-power fields (HPF) in this study. Main results and the role of chance The prevalence of CE with ≥5 CD138-positive cells in women with no intrauterine disorder, endometrial polyps, myomas, intrauterine adhesion (IUA), and septate uterus were 15.7%, 85.7%, 69.0%, 78.9%, and 46.2%, respectively. Multivariate analysis revealed that CE was diagnosed significantly more often in the endometrial polyp (p < 0.0001, odds ratio [OR] 27.69, 95% confidence interval [CI] 15.01–51.08) and IUA groups (p < 0.0001, OR 8.85, 95%CI 3.26–24.05). The CE recovery rates by hysteroscopic surgery in the women with endometrial polyps, myomas, IUA, and septate uterus were 89.7%, 100%, 92.8%, and 83.3%, respectively (p = 0.45). Limitations, reasons for caution This study has limitations. First, some patients received a combination of estrogen and progestin or dienogest before surgery. These medicines may influence the number of CD138-positive cells. Second, ≥5 CD138-positive cells per 10 HPF were defined as the diagnosis of CE; however, different diagnostic criteria may generate different results. Wider implications of the findings CE is primarily caused by intrauterine infection due to a wide variety of microorganisms; however, most CE cases with intrauterine abnormalities were cured by hysteroscopic surgery without antibiotic use. Most CE cases associated with intrauterine abnormalities may be noninfectious CE. Trial registration number not applicable

Associations of stem cell markers CD44, CD24 and ALDH1A1 with mammographic breast density in women with benign breast biopsies.

We examined associations of CD44, CD24 and ALDH1A1 breast stem cell markers with mammographic breast density (MBD), a well-established breast cancer (BCa) risk factor. We included 218 cancer-free women with biopsy-confirmed benign breast disease within the Nurses' Health Study (NHS) and NHSII. The data on BCa risk factors were obtained from biennial questionnaires. Immunohistochemistry (IHC) was done on tissue microarrays. For each core, the IHC expression was assessed using a semi-automated platform and expressed as percent of positively stained cells for each marker out of the total cell count. MBD was assessed with computer-assisted techniques. Generalised linear regression was used to examine the associations of each marker with square root-transformed percent density (PD), absolute dense and non-dense areas (NDA), adjusted for BCa risk factors. Stromal CD44 and ALDH1A1 expression was positively associated with PD (≥ 10% vs. <10% β = 0.56, 95% confidence interval [CI] [0.06; 1.07] and β = 0.81 [0.27; 1.34], respectively) and inversely associated with NDA (β per 10% increase = -0.17 [-0.34; -0.01] and β for ≥10% vs. <10% = -1.17 [-2.07; -0.28], respectively). Epithelial CD24 expression was inversely associated with PD (β per 10% increase = -0.14 [-0.28; -0.01]. Stromal and epithelial CD24 expression was positively associated with NDA (β per 10% increase = 0.35 [0.2 × 10-2; 0.70] and β per 10% increase = 0.34 [0.11; 0.57], respectively). Expression of stem cell markers is associated with MBD.

Phase 1/2 of EO2463 immunotherapy as monotherapy and in combination with lenalidomide and/or rituximab in indolent NHL (EONHL1-20/SIDNEY).

7058 Background: Follicular and marginal zone B cell lymphoma (FL; MZL) demonstrate an indolent course with heterogeneous outcomes anda potential for spontaneous remissions indicating immune system intervention. Therapeutic immunization is therefore an attractive approach but new antigens that evoke a strong immune response are needed. EO2463 expands pre-existing memory CD8+ T cells recognizing non-self protein sequences from gut bacteria which cross-react with B cell antigens and can kill HLA-A2 restricted cells (T2) loaded with target peptides. EO2463 includes 4 HLA-A2 synthetically produced epitopes which exhibit molecular mimicry with the B cell markers CD20, CD22, CD37, and CD268 (BAFF-receptor), as well as a the CD4 helper-epitope UCP2 derived from hTERT. EO2463 is being used to drive anti-tumor activity against B cell malignancies. Methods: Patients (pts) with FL and MZL, stage 1-3A, and HLA-A2, are eligible. In the safety lead-in pts with relapsed/refractory (R/R) disease were given EO2463 SC q2 weeks (w) x 4, then q4w, for a max of 12 months; at w7 lenalidomide (20 mg/day for 21/28 days up to 12 cycles) is added (EL), and if no complete remission (CR) at w19, rituximab (375 mg/m2 IV, q1w x 4, then q4w x 4) is also added (ER2). Doses evaluated: 150 μg and 300μg/peptide. Results: 3 pts received 150 μg/peptide and 6 pts 300 μg/peptide (EO2463 1 pat, EL 2 pts, ER2 6 pts): 2 MZL, 7 FL pts with median 2 lines (range 1-4) of prior systemic therapy. No related grade ≥3 adverse events were seen with EO2463 monotherapy. Most common related events were grade 1 to 2 local administration site reactions in 5/9 pts. Adverse events during combination treatment were as expected for R2. PET-CT on w6 after EO2463 monotherapy suggested clinical activity in 4/9 pts (1 PR, 1 pt size reduction 15%, 1 pt 20% reduced tracer uptake, 1 pt 5/6 target lesions reduced metabolic activity). Overall objective responses (OR) were seen in 6/9 pts (67%; 1st OR on EO2463 1 pat, EL 4 pts, ER2 1 pat), with CR in 5/9 (56%) pts; median time to response was 18w (range 8-43w), response ongoing in 5 pts, range 24-76w. Expansion of specific CD8+ T cells against mimic peptides and targeted B cell antigens were detected in all responding pts, incl. 2 pts with no measurable B cells at baseline (prior anti-CD20). Expansion of specific T cells was detected at w5 in 5/6 pts and was maintained as long as currently tested (up to w94, 43w after last EO2463 dose). No decline in expansions were seen after start rituximab. Conclusions: EO2463 (300 μg/peptide) monotherapy, EL, and ER2 are well tolerated, with encouraging clinical activity with EO2463 monotherapy and with subsequent CR in 5/9 pts on combo. Rapid and durable expansion of specific CD8+ T cells was seen in all responding pts, consistent with the preclinical hypothesis. Additional cohorts investigate EO2463 alone or in combination in newly diagnosed or R/R pts. Results will be reported at the meeting. Clinical trial information: NCT04669171 .

Preclinical efficacy and pharmacokinetics of an RNA-encoded T cell-engaging bispecific antibody targeting human claudin 6.

We present the preclinical pharmacology of BNT142, a lipid nanoparticle (LNP)-formulated RNA (RNA-LNP) encoding a T cell-engaging bispecific antibody that monovalently binds the T cell marker CD3 and bivalently binds claudin 6 (CLDN6), an oncofetal antigen that is absent from normal adult tissue but expressed on various solid tumors. Upon BNT142 RNA-LNP delivery in cell culture, mice, and cynomolgus monkeys, RNA is translated, followed by self-assembly into and secretion of the functional bispecific antibody RiboMab02.1. In vitro, RiboMab02.1 mediated CLDN6 target cell-specific activation and proliferation of T cells, and potent target cell killing. In mice and cynomolgus monkeys, intravenously administered BNT142 RNA-LNP maintained therapeutic serum concentrations of the encoded antibody. Concentrations of RNA-encoded RiboMab02.1 were maintained longer in circulation in mice than concentrations of directly injected, sequence-identical protein. Weekly injections of mice with BNT142 RNA-LNP in the 0.1- to 1-μg dose range were sufficient to eliminate CLDN6-positive subcutaneous human xenograft tumors and increase survival over controls. Tumor regression was associated with an influx of T cells and depletion of CLDN6-positive cells. BNT142 induced only transient and low cytokine production in CLDN6-positive tumor-bearing mice humanized with peripheral blood mononuclear cells (PBMCs). No signs of adverse effects from BNT142 RNA-LNP administration were observed in mice or cynomolgus monkeys. On the basis of these and other findings, a phase 1/2 first-in-human clinical trial has been initiated to assess the safety and preliminary efficacy of BNT142 RNA-LNP in patients with CLDN6-positive advanced solid tumors (NCT05262530).

Induction of FoxP3 Pre-mRNA Alternative Splicing to Enhance the Suppressive Activity of Regulatory T Cells from Amyotrophic Lateral Sclerosis Patients.

Forkhead box protein 3 (FoxP3) is a key transcription factor responsible for the development, maturation, and function of regulatory T cells (Tregs). The FoxP3 pre-mRNA is subject to alternative splicing, resulting in the translation of multiple splice variants. We have shown that Tregs from patients with amyotrophic lateral sclerosis (ALS) have reduced expression of full-length (FL) FoxP3, while other truncated splice variants are expressed predominantly. A correlation was observed between the reduced number of Tregs in the peripheral blood of ALS patients, reduced total FoxP3 mRNA, and reduced mRNA of its FL splice variant. Induction of FL FoxP3 was achieved using splice-switching oligonucleotides capable of base pairing with FoxP3 pre-mRNA and selectively modulating the inclusion of exons 2 and 7 in the mature mRNA. Selective expression of FL FoxP3 resulted in the induction of CD127low, CD152, and Helios-positive cells, while the cell markers CD4 and CD25 were not altered. Such Tregs had an increased proliferative activity and a higher frequency of cell divisions per day. The increased suppressive activity of Tregs with the induced FL FoxP3 splice variant was associated with the increased synthesis of the pro-apoptotic granzymes A and B, and perforin, IL-10, and IL-35, which are responsible for contact-independent suppression, and with the increased ability to suppress telomerase in target cells. The upregulation of Treg suppressive and proliferative activity using splice-switching oligonucleotides to induce the predominant expression of the FoxP3 FL variant is a promising approach for regenerative cell therapy in Treg-associated diseases.

Abstract PO5-06-06: Tumor/stromal expression of CD3/CD8/PD-1/PD-L1 and overall survival (OS) in patients (pts) with metastatic (met) HER2+ breast cancer (BC)

Abstract Background: The “Thousand Patient HER-2 database” project at Saint Vincent’s University Hospital (SVUH) Dublin contains a cohort of met HER2+ BC pts treated with trastuzumab, with OS ranging from short term to durable complete response (never relapsed). While the majority of pts progress on treatment, higher stromal tumor immune infiltrate has been associated with longer OS in met HER2+ BC. Using the SVUH database, we have identified a cohort of met HER2+ BC pts that received trastuzumab and have a broad OS range (0.3 months (mos) to 200.9 mos). This study examines the tumor and stromal levels of pan T cell marker CD3, cytotoxic T cell marker CD8, and immune checkpoints PD-L1 and PD-1 by immunohistochemistry (IHC) in primary and met samples from this cohort. Methods: Clinico-pathological data was available for formalin-fixed, paraffin-embedded biopsy specimens (n=45 primary, n=25 matched met biopsies). IHC staining was conducted for CD3 (Agilent IR50361-2), CD8 (Agilent IR62361-2), PD-L1, (Agilent M365329-2), and PD-1 (Roche 07099029001). A DAKO Link 48 Autostainer was utilised for staining, incorporating positive (tonsil tissue) and negative controls (isotype controls). Slide processing used an Aperio AT2 Digital Slide Scanner (Leica Biosystems), Aperio ImageScope 12.4 software (Leica Biosystems) and QuPath analysis software (University of Edinburgh). Following annotation of tumor areas, an algorithm was trained to identify immune cells and designate them as tumor or stromal. Data was expressed as the number of positively stained cells/mm2 tissue. A median cut-off was applied to denote “high” and “low” expression for CD3 and CD8. For PD-1 and PD-L1, samples with ≥ 1 stained cell/mm2 were designated as positive (pos) and samples with zero stained cells as negative (neg). The Kaplan Meier method was utilised for survival studies. A paired Student’s t test was utilised for primary vs metastatic site comparisons. Results: 51.1% of primary samples displayed high CD3 (23/45) and CD8 (23/45) expression in both the stromal and tumor compartments. Within the tumor compartment, 18.6% (8/43) of samples were pos for PD-L1 expression, and 57.8% (26/45) were pos for PD-1 expression. In the stromal compartment, 30.2% (13/43) of samples were pos for PD-L1 expression, and 68.9% (31/45) were pos for PD-1 expression. High tumor levels of CD3 (median OS CD3 high 113.4 mos vs CD3 low 7.9 mos, HR 0.2938 (95% CI 0.130-0.618), p=0.0012) and CD8 (median OS CD8 high 73.9 mos vs CD8 low 10.8 mos, HR 0.4764 (95% CI 0.238-0.954), p=0.0365), but not stromal levels of these markers, were associated with improved OS. There were no significant differences in CD3, CD8, PD-1 and PD-L1 levels between matched primary and met samples (p &amp;gt;0.05). Tumor, but not stromal, PD-L1 expression was associated with longer OS (HR 0.3120 (95% CI 0.146-0.667), p=0.0027). Interestingly, stromal PD-1 expression, but not tumor PD-1 expression, was also associated with longer OS (median OS PD-1 pos 64.7 mos vs. PD-1 neg 5.6 mos, HR 0.158 (95% CI 0.060-0.411), p=0.0002). Conclusion: Our results report a link between OS and tumor (CD3+, CD8+ and PD-L1+)/stromal (PD-1+) immune cell infiltrate in met HER2+ BC patients treated with trastuzumab. Further expansion of this preliminary dataset is warranted. Citation Format: Denis Collins, Janet McCormack, Laura Ivers, Jose Javier Berenguer-Pina, Jo Ballot, Cecily Quinn, Darko Skrobo, Alex Eustace, Naomi Walsh, Aurelie Fabre, John Crown. Tumor/stromal expression of CD3/CD8/PD-1/PD-L1 and overall survival (OS) in patients (pts) with metastatic (met) HER2+ breast cancer (BC) [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 PO5-06-06.

Associations of Early-Life and Adult Anthropometric Measures with the Expression of Stem Cell Markers CD44, CD24, and ALDH1A1 in Women with Benign Breast Biopsies.

According to the stem cell hypothesis, breast carcinogenesis may be related to the breast stem cell pool size. However, little is known about associations of breast cancer risk factors, such as anthropometric measures, with the expression of stem cell markers in noncancerous breast tissue. The analysis included 414 women with biopsy-confirmed benign breast disease in the Nurses' Health Study and Nurses' Health Study II. Birthweight, weight at age 18, current weight, and current height were reported via self-administered questionnaires. IHC staining of stem cell markers (CD44, CD24, and aldehyde dehydrogenase family 1 member A1) in histopathologically normal epithelial and stromal breast tissue was quantified using an automated computational image analysis system. Linear regression was used to examine the associations of early-life and adult anthropometric measures with log-transformed stem cell marker expression, adjusting for potential confounders. Birthweight [≥10.0 vs. <5.5 lbs: β (95% confidence interval) = 4.29 (1.02, 7.56); P trend = 0.001 in the stroma] and adult height [≥67.0 vs. <63.0 inch: 0.86 (0.14, 1.58); P trend = 0.02 in the epithelium and stroma combined] were positively associated with CD44 expression. Childhood body fatness was inversely associated (P trend = 0.03) whereas adult height was positively associated with CD24 expression in combined stroma and epithelium (P trend = 0.03). Our data suggest that anthropometric measures, such as birthweight, adult height, and childhood body fatness, may be associated with the stem cell expression among women with benign breast disease. Anthropometric measures, such as birthweight, height, and childhood body fatness, may have long-term impacts on stem cell population in the breast.

Abstract 5520: β2 adrenergic receptor signaling promotes colorectal cancer liver metastasis

Abstract Liver is the most common site for cancer metastases, which may be attributed to the hepatic immunosuppressive microenvironment. Recent studies have highlighted the immunomodulatory and tumor-supportive roles of neurotransmitter-receptor signals in various cancers. Given that various neurotransmitter receptors are expressed in the liver, we aimed to investigate their possible involvement in the metastatic-prone liver microenvironment. Single-cell RNA sequencing analysis identified ADRB2, which encodes β2-adrenergic receptor (β2-AR), as the top-upregulated neurotransmitter receptor gene in colorectal cancer (CRC) and liver metastasis in patients with CRC liver metastasis (CRLM). Co-immunofluorescence (co-IF) staining confirmed the colocalization of β2-AR with myeloid cell marker CD11b instead of T cell marker CD3 and NK cell marker CD56 in patients with CRLM. Consistently, we observed the upregulation of CD11b/β2-AR double-positive cells in the liver tissues from mice bearing CRC or CRLM compared to naïve mice through co-IF staining. High-dimensional flow cytometry analysis further revealed that β2-AR was mainly expressed in macrophages and myeloid-derived suppressor cells (MDSCs). Treatment of CRC-tumor bearing mice with pan β-AR inhibitor propranolol and β2-AR specific inhibitor showed a potential to reduce liver metastatic incidence without suppressing primary CRC. Mechanistically, β2-AR activation may upregulate the expression of S100a8/9 to enhance the immunosuppressive activity of macrophages and MDSCs, thereby promoting CRC liver metastasis. Taken together, our findings reveal the potential of targeting β2-AR signaling for the prevention and treatment of liver metastasis. This project is supported by the Hong Kong University Grants Committee through the General Research Fund (14104820, 14107622, 24110323) and the Strategic Seed Funding for Collaborative Research Scheme (SSFCRS) from the Chinese University of Hong Kong. Conflict of interest: The authors declare no conflict of interest. Citation Format: Yixuan Zhang, Jintian Chen, Thomas Ting Hei Chan, Huanyu Wang, Jiahuan Lu, Jingqing Li, Jingying Zhou. β2 adrenergic receptor signaling promotes colorectal cancer liver metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5520.

Abstract 2916: Discoidin domain receptor 1 (DDR1) expression is associated with degree of immune exclusion across epithelial tumors

Abstract Background: Discoidin domain receptor 1 (DDR1) is highly expressed in epithelial cancers and has been implicated in tumor growth, invasion, and lack of response to therapy. DDR1 contributes to immune exclusion by promoting tumor collagen alignment in in vivo models. However, it is unclear how DDR1 expression impacts immune cell infiltration in human tumors. A first-in-human trial of PRTH-101, a DDR1-targeted therapeutic antibody, is underway. Establishing a correlation between DDR1 expression and immune infiltration in the tumor microenvironment (TME) will shed light on the role of DDR1 in the TME and inform indication and patient selection strategies for DDR1-targeted therapies. Methods: Adjacent formalin fixed paraffin embedded slides from colorectal (CRC), non-small cell lung (NSCLC), ovarian (OC), pancreatic (PDAC), and triple-negative breast cancers (TNBC) were stained by H&amp;E and a multiplex immunofluorescence (mIF) panel containing DDR1 and immune cell markers CD8 and CD45. Tumor-stromal segmentation and cell identification was done from H&amp;E using AI-powered models developed by PathAI (PathExplore™) or from mIF by image analysis using QuPath. DDR1 mRNA expression was measured by bulk RNA-sequencing, while DDR1 protein expression was measured by mIF. Immune Exclusion Scores (IESs) were calculated for each tumor based on lymphocyte density (from H&amp;E), CD8+ T cell density (from mIF), or CD45+ cell density (from mIF). Each IES is the orthogonal distance between the coordinate of immune cell density in tumor epithelium and stroma, and the regression line representing equal density in epithelium and stroma. Correlations between lymphocyte, CD8, and CD45 IESs and DDR1 mRNA or protein expression levels were evaluated. Results: Both DDR1 mRNA and protein expression levels were significantly correlated with all Immune Exclusion Scores (IESs) measured by the distribution of lymphocytes (H&amp;E), CD8+, or CD45+ cells (mIF) in tumor epithelium and stroma (R: 0.31-0.55, adjusted p: &amp;lt;0.001-0.01) at a pan-cancer level. While the degree of the correlation varied between tumor indications by IES immune cell type, pancreatic cancer exhibited the strongest correlation between the lymphocyte-based IES and DDR1 mRNA and protein expression (R: 0.48-0.54, adjusted p: 0.04-0.07). DDR1 mRNA was also moderately correlated with lymphocyte IES in NSCLC and TNBC (R: 0.37-0.48, adjusted p: 0.12-0.14). Conclusions: We developed a continuous scoring method to quantify the degree of immune exclusion in tumors based on the spatial distributions of lymphocytes, CD8+ T cells, and CD45+ immune cells from H&amp;E and mIF images. DDR1 mRNA and protein expression are correlated with immune exclusion at both the pan-cancer and specific indication level. This adds additional insight into the role of DDR1 in human cancers and may be useful in selecting indications and stratifying patients for DDR1-targeted therapies. Citation Format: Xinwei Sher, Fredrick D. Gootkind, Thomas Schürpf, Florent Peyraud, Jean-Philippe Guégan, Antoine Italiano, G. Travis Clifton, Laura A. Dillon. Discoidin domain receptor 1 (DDR1) expression is associated with degree of immune exclusion across epithelial tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2916.

Associations of reproductive breast cancer risk factors with expression of stem cell markers in benign breast tissue.

We investigated the associations of reproductive factors known to influence breast cancer risk with the expression of breast stem cell markers CD44, CD24, and ALDH1A1 in benign breast biopsy samples. We included 439 cancer-free women with biopsy-confirmed benign breast disease within the Nurses' Health Study (NHS) and NHSII. The data on reproductive and other breast cancer risk factors were obtained from biennial questionnaires. Immunohistochemistry (IHC) was performed on tissue microarrays. For each core, the IHC expression was assessed using a semi-automated platform and expressed as % of cells that stained positive for a specific marker out of the total cell count. Generalized linear regression was used to examine the associations of reproductive factors with a log-transformed expression of each marker (in epithelium and stroma), adjusted for other breast cancer risk factors. In multivariate analysis, the time between menarche and age at first birth was inversely associated with CD44 in epithelium (β per 5 years = -0.38, 95% CI -0.69; -0.06). Age at first birth and the time between menarche and age at first birth were inversely associated with ALDH1A1 (stroma: β per 5 years = -0.43, 95% CI -0.76; -0.10 and β = -0.47, 95% CI -0.79; -0.15, respectively; epithelium: β = -0.15, 95% CI -0.30; -0.01 and β = -0.17, 95% CI -0.30; -0.03, respectively). Time since last pregnancy was inversely associated with stromal ALDH1A1 (β per 5 years = -0.55, 95% CI -0.98; -0.11). No associations were found for CD24. The observed associations were similar in premenopausal women. In postmenopausal women, lifetime duration of breastfeeding was inversely associated with stromal ALDH1A1 expression (β for ≥24 vs. 0 to <1 months = -2.24, 95% CI 3.96; -0.51, p-trend = 0.01). Early-life reproductive factors may influence CD44 and ALDH1A1 expression in benign breast tissue.

Abstract B007: Establishment of patient-derived brain metastasis initiating cells as a novel preclinical model of breast cancer metastatization

Abstract Brain metastasis from breast cancer (BCBM) are lethal tumors occurring in 10-15% of IV stage breast cancers against which only limited therapeutic options are available. A critical hindrance to the design of novel therapeutic approaches is the narrow set of preclinical models to study BCBM pathophysiology, chiefly those resembling the stem-like population which is thought be the main source of BCBM dissemination and progression. We aimed to isolate patient-derived metastasis-initiating cells (MICs) to furnish novel preclinical models of metastatization. MICs were cultured as stem-enriching tumorspheres, which we characterized in term of i) brain/breast/stem markers expression ii) tumorigenicity, iii) growth and in vitro/in vivo self-renewal, iv) adhesion to organ-specific endothelium, v) metastatic potential and vi) drug responsiveness. Further, we transcriptionally profiled MIC-driven organ-specific metastasis. Patient-derived MICs expressed epithelial markers while the expression of brain markers was poor, coherently with their epithelial origins. About 90% of MICs were positive to the expression of the putative stem cell markers CD15, CD24 and CD44, while CD133 and ALDH1A1 were less represented. MICs were grown indefinitely in stem cell medium and successfully formed spheres in serial clonogenic assays, suggesting their endowment with stem-like traits. Similarly, their ability to form tumors when orthotopically injected in mice brain was maintained upon serial transplantation and under in vivo limiting dilution conditions. The representative PR60 MICs showed metastatic potential to mice brain and bones upon either intracardiac or intranipple transplantation. Of note, independently of the route of injection, this metastatization pattern wholly mirrored the clinical course of the patient they had been derived from. PR60-driven brain metastasis also recapitulated patient tumor histology, cytoarchitecture and marker expression. Further confirming their brain-tropism, PR60 MICs preferentially adhered to human brain compared to non-brain endothelium under shear stress mimicking in vivo hemodynamic conditions. Transcriptomically, PR60 MICs displayed high correlation with the BCBM of origin but retain the ability to form intra-nipple tumors resembling patient primitive breast tumor. PR60-derived brain and bone metastasis reveal a distinct expression profile suggesting that PR60 MICs can colonize different organs rewiring their transcriptomic landscape. Compared to the whole tumor of origin, MICs revealed an up-regulation of drug resistance signatures, which are often linked with stem-like traits, and that were functionally validated by a high throughput drug screening. Altogether, we established a robust and versatile MIC model that reliably mirrors the brain metastasis of origin and mimics the different steps of brain metastatic dissemination for in vitro and in vivo studies. This model has the potential to provide further knowledge for future therapy development for BCBM management. Citation Format: Stefania Faletti, Cristina Richichi, Daniela Osti, Elena Ceccacci, Massimiliano Del Bene, Camilla Cerutti, Giovanni Bertalot, Monica Patanè, Bianca Pollo, Giuliana Pelicci. Establishment of patient-derived brain metastasis initiating cells as a novel preclinical model of breast cancer metastatization [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr B007.

Expression of CD25, mast cell markers and T-cell markers in eosinophilic esophagitis

While eosinophilic esophagitis (EOE) is defined by histologic presence of eosinophils, a few studies have established the presence of mast cells in EOE and even shown their correlation with symptom persistence despite resolution of eosinophils. Expression of aberrant mast cell markers CD25 and CD2 have not been studied in EOE. This study quantifies the number of hotspot cells per high power field expressing CKIT/CD117, tryptase, CD25, CD2 and CD3 by immunohistochemical stains in endoscopic esophageal biopsies of the following three cohorts: (1) established and histologically confirmed EOE, (2) suspected EOE with biopsies negative for eosinophils, and (3) no history of or suspicion for EOE with histologically unremarkable biopsies. In this study, mast cells were highlighted by CKIT and tryptase in EOE, and not seen in other clinically mimicking cases. There were also significantly higher densities of CD25 and pan-T-cell marker staining in EOE cases. These findings suggest an inflammatory cellular milieu in EOE, beyond just eosinophils, that can be demonstrated by immunohistochemistry, and that invite further study into the role that these cells may play in EOE.

Study on the Mechanism of Notch Pathway Mediates the Role of Lenvatinib-resistant Hepatocellular Carcinoma Based on Organoids.

The emergence of treatment resistance has hindered the efficacy of targeted therapies used to treat patients with hepatocellular carcinoma (HCC). This study aimed to explore the mechanism of organoids constructed from lenvatinib-resistant HCC cells. Hep3B cell and human HCC organoids were cultured and identified using hematoxylin and eosin staining and Immunohistochemistry. Lenvatinib-sensitive/ resistant Hep3B cells were constructed using lenvatinib (0, 0.1, 1, and 10 μM) and lenvatinib (0, 1, 10, and 100 μM). qRT-PCR and flow cytometry were utilized to determine HCC stem cell markers CD44, CD90, and CD133 expressions. Transcriptome sequencing was performed on organoids.-Western blot evaluated Notch pathwayrelated proteins (NOTCH1 and Jagged) expressions. Furthermore, DAPT, an inhibitor of the Notch pathway, was used to investigate the effects of lenvatinib on resistance or stemness in organoids and human HCC tissues. The organoids were successfully cultivated. With the increase of lenvatinib concentration, sensitive cell organoids were markedly degraded and ATP activity was gradually decreased, while there was no significant change in ATP activity of resistant cell organoids. CD44 expressions were elevated after lenvatinib treatment compared with the control group. KEGG showed that lenvatinib treatment of organoids constructed from Hep3B cells mainly activated the Notch pathway. Compared with the control group, NOTCH1 and Jagged expressions elevated, and ATP activity decreased after lenvatinib treatment. However, ATP activity was notably decreased after DAPT treatment. Moreover, DAPT inhibited lenvatinib resistance and the increase in the expressions of CD44 caused by lenvatinib. Besides, 100 μM lenvatinib significantly inhibited the growth and ATP activity of human HCC organoids, and DAPT increased the inhibitory effect of lenvatinib. Lenvatinib regulated resistance and stemness in organoids via the Notch pathway.

Single institution study of the immune landscape for canine oral melanoma based on transcriptome analysis of the primary tumor.

Understanding a tumor's immune context is paramount in the fight against cancer. Oral melanoma in dogs serves as an excellent translational model for human immunotherapy. However, additional study is necessary to comprehend the immune landscape of dog oral melanomas, including their similarity to human melanomas in this context. This retrospective study utilizes formalin-fixed paraffin-embedded (FFPE) tissue samples to analyze RNA sequences associated with oral melanoma in dogs. Nanostring Technologies was used for conducting RNA sequencing. The focus is on understanding the differences between melanoma tumors restricted to the oral cavity (OL) and the same primary oral tumors with a history of metastasis to the lymph nodes or other organs (OM). Normal buccal mucosa samples are also included as a normal tissue reference. In the OM patient group, gene signatures exhibit significant changes relative to the OL patient group, including significantly decreased expression of S100, BRAF, CEACAM1, BCL2, ANXA1, and tumor suppressor genes (TP63). Relative to the OL tumors, the OM tumors had significantly increased expression of hypoxia-related genes (VEGFA expression), cell mobility genes (MCAM), and PTGS2 (COX2). The analysis of the immune landscape in the OM group indicates a shift from a possible "hot" tumor suppressed by immune checkpoints (PDL1) to significantly heightened expression not only of those checkpoints but also the inclusion of other immune blockades such as PD1 and IDO2. In addition, the OM group had significantly reduced expression of Toll-like receptors (TLR4) and IL-18 relative to the OL group, contributing to the tumor's immune escape. Additionally, signs of immune cell exhaustion are evident in both the OM and OL groups through significantly increased expression of TIGIT relative to normal tissue. Both the OM and OL groups had significantly increased expression of the immune cell marker CD4 expression relative to normal tissue. Further, CD4 expression significantly decreased in OM relative to OL; however, this study cannot determine the specific cell types expressing CD4 in OM and OL tumors. This preliminary study reports significant changes in gene expression for oral melanoma between canine patients with localized disease relative to those with metastatic disease. In the future, a more in-depth investigation involving immunohistochemistry analysis and single-cell RNA expression is necessary to confirm our findings.

REACTIVATION OF THE NUCLEUS PULPOUS CELLS BY PLATELET DERIVATIVES AS A NEW TOOL IN INTERVERTEBRAL DISC REGENERATION

Degenerative disc disease, associated to low back pain, afflicts more than 50% of humans, and represents a major healthcare problem, especially for the pathology initiation. Current treatments range from conservative strategies to more invasive surgical techniques, such as disc removal and vertebral fusion. In the Intervertebral Disease (IVD) the nucleus pulposus (NP) degeneration is a key factor for the pathology initiation. Several tissue engineering approaches aiming to restore the appropriate NP cell (NPCs) and matrix content, were attempted by using adult stromal cells either from bone marrow or adipose tissue, chondrocytes, notochordal cells and more recently also pluripotent stem cells. However, none was fully satisfactory since the NP acid and a-vascularized environment appeared averse to the implanted heterologous cells. Several studies demonstrated the efficacy of platelet derivatives such as platelet rich plasma (PRP) in promoting the regeneration of connective tissues. We investigated the efficacy of PRP on NPCs proliferation and differentiation with the goal to propose the direct stimulation of resident cells (stimulation of endogenous cells – less invasive surgical procedure) or the implantation of NPCs expanded in vitro in the presence of PRP as therapeutic agents in IVD degeneration.NPCs were isolated from small fragments of NP explants, cultivated in medium supplemented with PRP or FCS (standard condition control) and characterized by FACS analysis for the expression of the typical mesenchymal stem cells markers CD34, CD44, CD45, CD73, CD90 and CD105. NPCs cultured in PL showed a phenotypic profile like the cells cultured in FCS. However, compared to NPCs expanded in the presence of FCS, NPCs expanded in PRP showed a much better proliferation and differentiation capacity. NPCs differentiation was evaluated by the cell ability to produce an organized metachromatic cartilaginous matrix, confirmed by the positive immunohistochemical staining for chondrogenic markers.

Características fenotípicas y potencial de diferenciación de las células madre mesenquimales gingivales en la hiperglucemia: un estudio exploratorio ex vivo

Background: The therapeutic use of gingival mesenchymal stem cells (GMSCs) as autologous cells may pose the challenge of alterations inflicted by the hyperglycemic environment. Objective: This study aims to assess the effects of hyperglycemia on the characteristics of GMSCs in diabetics. Materials and Methods: 10 patients who consented and fulfilled the criteria for inclusion and exclusion were recruited and categorized as test (HbA1c &gt; 6.5) and control (HbA1c &lt; 6.0). Gingival explants were obtained from gingival collar of teeth, washed, digested and cultured. The cells were subjected to microscopic observation to assess phenotype characteristics, and flow cytometry and qRT-PCR to assess differentiation potential. Stem cell markers CD90, CD73, CD105, CD34, CD45, HLA DR &amp; HLA ABC, osteogenic differentiation markers RUNX2 &amp; OCN, adipogenic differentiation markers PPARG2 &amp; FABP4 and chondrogenic differentiation markers SOX9 &amp; AGCN were evaluated. Results: Microscopic appearance of spindle shaped cells was found to be comparable in both groups. Flow cytometry results demonstrated comparable expressions with both groups, samples being positive for CD90, CD73, CD105, HLA ABC and negative for CD34, CD45 &amp; HLA DR. There were variations in the expression of markers when assessed for differentiation potentials. Conclusions: The hyperglycemic environment did not manifest any changes in the phenotypic characteristics of GMSCs among diabetics. However, the expression of certain differentiation markers was significantly altered in the diabetic test population included. Further research is being conducted to understand the GMSCs in a hyperglycemic environment with an aim to develop strategies to optimize its clinical implications.