Fraction Of Positive Cells Research Articles

Abstract B008: High expression of the exhaustion markers PD1 and PD-L1 in non-muscle invasive bladder cancer is associated with poor outcome following Bacillus Calmette-Guérin immunotherapy

Abstract Introduction: The recommended treatment for high-risk non-muscle invasive bladder cancer (NMIBC) is intravesical instillations of Bacillus Calmette-Guérin (BCG). However, 40 % experience recurrence within 5 years despite completing BCG treatment. T cell exhaustion has been associated with poor outcome following treatment with BCG. Here we investigated if T cell exhaustion, characterized by protein expression of PD1 and PD-L1 measured in paired samples obtained before and after BCG treatment could further explain BCG response and help predict outcome in patients with NMIBC. Methods: We included 111 patients with NMIBC, from which we had 183 TURB-T samples obtained before and after BCG treatment. Using immunohistochemistry (IHC) staining of sections from tissue microarrays (TMAs) with triplicate core biopsies, we investigated the protein expression of the exhaustion markers PD1 (clone EP239) and PD-L1 (clone 22c3). Data was analyzed using digital pathology software (Visiopharm®), where tissue areas were defined as either carcinoma or stroma based on PanCK staining. Cells were scored as positive when detection of a nucleus was combined with a positive PD1 or PD-L1 stain, which was based on visually defined thresholds to differentiate between positive and unspecific staining. In total, 167 samples from 105 patients were considered suitable for further analysis (cell count > 200 in carcinoma and stromal areas) with 2-3 cores per sample, resulting in a total of 441 tissue cores. The average cell count was 5732 per core (defined by positive nuclei stain). Stratification of samples into high or low expression was based on a median split of the positive cell fraction. Results: When investigating the number of PD1 and PD-L1 positive cells in the tumors (carcinoma and stroma combined), we observed that PD-1 expression significantly increased with tumor stage in pre- (p=0.002) and post-BCG (p=0.006) tumor samples. PD1 expression was also increased in high-grade tumors compared to low-grade tumors in pre-BCG samples (p = 0.001). Furthermore, we observed a significant association between tumors of higher stage and high PD-L1 expression in the pre-BCG samples (p = 0.007). Patients with low expression of PD1 and PD-L1 in the pre-BCG tumor samples had a superior high-grade recurrence-free survival (HG-RFS) compared to patients with high PD1 (p=0.002) and PD-L1 (p=0.021) expression. In addition, low expression of PD-L1 in pre-BCG tumors was correlated with a better progression-free survival compared to the patients with high PD-L1 expression (p = 0.013). Conclusion: Protein expression of PD1 and PD-L1 in pre-BCG tumor samples were correlated to higher stage and grade as well as worse HGFRS, indicating that T cell exhaustion plays an important role in resistance to BCG treatment. Citation Format: Tine G. Andreasen, Trine Strandgaard, Jørgen B. Jensen, Lars Dyrskjøt. High expression of the exhaustion markers PD1 and PD-L1 in non-muscle invasive bladder cancer is associated with poor outcome following Bacillus Calmette-Guérin immunotherapy [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr B008.

EZH2 Expression in Head-and-Neck Squamous Cell Cancer in Young Patients.

EZH2 (Enhancer of zeste homolog 2) promotes tumor growth and survival through numerous mechanisms and is a promising target for novel therapeutic approaches. We aimed to characterize the expression of EZH2 in the tumors of young head-and-neck squamous cell cancer (HNSCC) patients in comparison with the general HNSCC patient population. We used formalin-fixed, paraffin-embedded tissue blocks from 68 random young HNSCC patients (≤39 years, median age: 36 years; diagnosed between 2000 and 2018), which were compared with the samples of 58 age- and gender-matched general HNSCC subjects (median age: 62 years; all diagnosed in the year 2014). EZH2 and p53 expression of the tumors was detected using immunohistochemical staining. Lower EZH2 expression was found to be characteristic of the tumors of young HNSCC patients as opposed to the general population (median EZH2 staining intensity: 1 vs. 1.5 respectively, p < 0.001; median fraction of EZH2 positive tumor cells: 40% vs. 60%, respectively, p = 0.003, Mann-Whitney). Cox analysis identified a more advanced T status (T3-4 vs. T1-2), a positive nodal status, and alcohol consumption, but neither intratumoral EZH2 nor p53 were identified as predictors of mortality in the young patient group. The lower EZH2 expression of young HNSCC patients' tumors discourages speculations of a more malignant phenotype of early-onset tumors and suggests the dominant role of patient characteristics. Furthermore, our results might indicate the possibility of an altered efficacy of the novel anti-EZH2 therapies in this patient subgroup.

Analysis of Metabolic Mechanism of CD19CAR-T with CD79A/CD40 Co-Stimulatory Domain Based on Comparison with Established CAR-Ts

Introduction: Anti-CD19 chimeric antigen receptor T cell (CD19CAR-T) therapy has achieved significant clinical outcomes and changed treatment strategy in B-cell malignancies. However, recurrence after CAR-T therapy remains a major obstacle due to poor peak expansion and short persistence of CAR-T cells. We have reported that CD19CAR-T incorporating a co-stimulatory domain consisting of CD79A and CD40 (79A40CAR) provided superior anti-tumor efficacy and survival compared to CD19CAR-T with CD28 or 4-1BB (CD28CAR/4-1BBCAR) in mice xenograft models. 79A40CAR exhibited sustained NF-κB phosphorylation and superior in vivo efficacy compared to the CD28CAR or 4-1BBCAR. (Molecular Therapy 2021, Julamanee J et al.) In this study, we aimed to clarify the molecular mechanism of 79A40CAR by comparing with CD28/4-1BBCAR in terms of T-cell metabolism. Methods: CD8+T cells were isolated from PBMCs of healthy donors and retrovirally transduced with either CD28IC-, 4-1BBIC-, or 79A40IC-CD19CARs. CD28CAR, 4-1BBCAR, or 79A40CAR were cocultured with 100 Gy irradiated NALM6 at E:T ratio = 1:5, respectively. Repeated antigen stimulations (Ag-stim) were performed, and day 7 after each stimulation was subjected to analysis. RNA-seq, expansion capacity, T-cell phenotype, and metabolic evaluation using a seahorse flux analyzer (Agilent Technologies) were performed. After Ag-stim, CD45RA negative CD62L positive and CD45RA negative CD62L positive CAR-T cell fractions were sorted as central memory (T CM) and effector memory (T EM), respectively. T CM and T EM received Ag-stim. After Ag-stim, we performed evaluation of mitochondrial functionality with MitoTracker based on mitochondrial mass and membrane potential. Results: All CD19CAR genes were successfully transduced into primary CD8+ T-cells. To understand the differences between co-stimulatory domains, we first confirmed bulk RNA-seq, expansion capacity, and T-cell phenotype. After Ag-stim, 79A40CAR showed enhancement of the NF-κB pathway. We detected few DEGs when 79A40CAR compared to 4-1BBCAR, however, we detected 162 upregulated DEGs when 79A40CAR compared to CD28CAR. Principal component analysis of gene expression demonstrated similarity between 4-1BBCAR and 79A40CAR. We confirmed that 79A40CAR had superior expansion capacity than CD28, or 4-1BBCAR after repeated Ag-stim. PD-1 and Tim-3 expression in 79A40CAR was lowest after repeated Ag-stim. In the evaluation of T-cell differentiation, 4-1BB and 79A40CAR showed an earlier transition to the effector phenotype compared to CD28CAR after Ag-stim. To evaluate differences in terms of T-cell metabolism, we performed metabolic analysis using a seahorse flux analyzer. After repeated Ag-stim, 4-1BB and 79A40CAR showed a predominant oxidative phosphorylation metabolic pathway (Fig1A) and higher spare respiratory capacity (SRC), which has been correlated with mitochondrial functionality (Fig1B), than CD28CAR. After repeated Ag-stim, 4-1BB and 79A40CAR demonstrated higher ECAR than CD28CAR. Because we observed differences in the expression of T-cell differentiation phenotype, we performed cell sorting to evaluate T-cell metabolism of each differentiation phenotype. After applying repeated Ag-stim to T CM and T EM-derived CD19CAR-T, mitochondrial functionality was evaluated using Mitotracker. T CM- (Fig1C) and T EM- (Fig1D) derived 4-1BB and 79A40CAR had more functional mitochondria and fewer dysfunctional mitochondria compared to CD28CAR. Conclusion: 79A40CAR seems to preserve the mitochondrial oxidative phosphorylation-dominant metabolic pathway and supply energy demands for a longer period, especially when compared to CD28CAR. 79A40CAR showed functionally very similar to 4-1BBCAR. We showed that CD19CAR-Ts had different metabolic pathway according to the different co-stimulatory domains. To reveal additional metabolic pathway differences between 4-1BBCAR and 79A40CAR, we are now conducting proteomics analysis.

SIX1 as a Novel Immunohistochemical Marker in the Differential Diagnosis of Rhabdomyosarcoma

Background: Differential diagnosis of rhabdomyosarcoma (RMS) is challenging. Sineoculis homeobox homolog 1 (SIX1) is an oncogene involved in skeletal muscle differentiation. We compared protein expression patterns of SIX1 in RMS and its most common differential diagnoses. Methods: SIX1 immunohistochemistry in 36 RMS and in 33 tumors from seven differential diagnostic subtypes were evaluated. The fraction of SIX1 positive tumor cells was scored by three independent observers. Results: A majority (75%) of the evaluated RMS expressed SIX1 in at least 50% of tumor cells and all except one RMS had more than 25% positive tumor cells. Neuroblastoma had less than 1% SIX1 positive tumor cells. Gonadoblastoma, malignant rhabdoid tumor, and Ewing sarcoma had 10% or less positive tumor cells. Pleuropulmonary blastoma exhibited 26–50% positive tumor cells and synovial sarcoma >50% positive cells. Conclusion: SIX1 immunohistochemistry is positive in most RMS, and occasionally in some tumors within the differential diagnoses of RMS.

Evaluation of role of serum prolactin &amp; ki-67 in different stages of breast cancer

Breast cancer is the most frequent cancer among women. Prolactin (PRL) is a hormone secreted from anterior pituitary gland which stimulates cell proliferation, survival of cancer cells, its migration, invasion and angiogenesis. Breast cancer is strongly associated with raised plasma Prolactin level (PRL). The Prolactin behaves like Growth Hormone and its actions by the growth-promoting JAK/STAT pathway suggests its tumor-promoting effects.Ki-67 a Nuclear Protein is highest in Luminal B, Ki67 are not related to age but is correlated with tumor size. Fraction of Ki-67 positive tumor cells correlates with the clinical course of cancer cells. Hence, Ki- 67 can be an effective target in cancer therapy. Ki-67% and Serum Prolactin level may be considered a valuable biomarker in breast cancer patients and be used in treatment, follow-up and Prognosis.

Postnatal Osterix but not DMP1 lineage cells significantly contribute to intramembranous ossification in three preclinical models of bone injury.

Murine models of long-bone fracture, stress fracture, and cortical defect are used to discern the cellular and molecular mediators of intramembranous and endochondral bone healing. Previous work has shown that Osterix (Osx+) and Dentin Matrix Protein-1 (DMP1+) lineage cells and their progeny contribute to injury-induced woven bone formation during femoral fracture, ulnar stress fracture, and tibial cortical defect repair. However, the contribution of pre-existing versus newly-derived Osx+ and DMP1+ lineage cells in these murine models of bone injury is unclear. We addressed this knowledge gap by using male and female 12-week-old, tamoxifen-inducible Osx Cre_ERT2 and DMP1 Cre_ERT2 mice harboring the Ai9 TdTomato reporter allele. To trace pre-existing Osx+ and DMP1+ lineage cells, tamoxifen (TMX: 100mg/kg gavage) was given in a pulse manner (three doses, 4weeks before injury), while to label pre-existing and newly-derived lineage Osx+ and DMP1+ cells, TMX was first given 2weeks before injury and continuously (twice weekly) throughout healing. TdTomato positive (TdT+) cell area and cell fraction were quantified from frozen histological sections of injured and uninjured contralateral samples at times corresponding with active woven bone formation in each model. We found that in uninjured cortical bone tissue, Osx Cre_ERT2 was more efficient than DMP1 Cre_ERT2 at labeling the periosteal and endosteal surfaces, as well as intracortical osteocytes. Pulse-labeling revealed that pre-existing Osx+ lineage and their progeny, but not pre-existing DMP1+ lineage cells and their progeny, significantly contributed to woven bone formation in all three injury models. In particular, these pre-existing Osx+ lineage cells mainly lined new woven bone surfaces and became embedded as osteocytes. In contrast, with continuous dosing, both Osx+ and DMP1+ lineage cells and their progeny contributed to intramembranous woven bone formation, with higher TdT+ tissue area and cell fraction in Osx+ lineage versus DMP1+ lineage calluses (femoral fracture and ulnar stress fracture). Similarly, Osx+ and DMP1+ lineage cells and their progeny significantly contributed to endochondral callus regions with continuous dosing only, with higher TdT+ chondrocyte fraction in Osx+ versus DMP1+ cell lineages. In summary, pre-existing Osx+ but not DMP1+ lineage cells and their progeny make up a significant amount of woven bone cells (particularly osteocytes) across three preclinical models of bone injury. Therefore, Osx+ cell lineage modulation may prove to be an effective therapy to enhance bone regeneration.

Epithelial and Stromal Characteristics of Primary Tumors Predict the Bone Metastatic Subtype of Prostate Cancer and Patient Survival after Androgen-Deprivation Therapy.

Prostate cancer (PC) bone metastases can be divided into transcriptomic subtypes, by us termed MetA-C. The MetB subtype, constituting about 20% of the cases, is characterized by high cell cycle activity, low androgen receptor (AR) activity, and a limited response to standard androgen deprivation therapy (ADT). Complementary treatments should preferably be introduced early on if the risk of developing metastases of the MetB subtype is predicted to behigh. In this study, we therefore examined if the bone metastatic subtype and patient outcome after ADT could be predicted by immunohistochemical analysis of epithelial and stromal cell markers in primary tumor biopsies obtained at diagnosis (n = 98). In this advanced patient group, primary tumor International Society of Urological Pathology (ISUP) grade was not associated with outcome or metastasis subtype. In contrast, high tumor cell Ki67 labeling (proliferation) in combination with low tumor cell immunoreactivity for PSA, and a low fraction of AR positive stroma cells in the primary tumors were prognostic for poor survival after ADT. Accordingly, the same tissue markers were associated with developing metastases enriched for the aggressive MetB subtype. The development of the contrasting MetA subtype, showing the best response to ADT, could be predicted by the opposite staining pattern. We conclude that outcome after ADT and metastasis subtype can, at least to some extent, be predicted by analysis of primary tumor characteristics, such as tumor cell proliferation and PSA expression, and AR expression in stromal cells.

Prognostic impact of stromal periostin expression in upper urinary tract urothelial carcinoma

BackgroundPeriostin is an extracellular matrix protein that has been known to be implicated in fibrillogenesis and cell migration, including cancer metastasis. Periostin overexpression in cancer cells and/or intervening stroma is usually related to tumor progression and poor patient outcomes in various human cancers; however, its role in urothelial carcinoma, especially upper urinary tract urothelial carcinomas (UTUCs), remains inconclusive.MethodsSamples from 126 consecutive cases of invasive UTUC (69 renal pelvic cancers and 57 ureteral cancers) were histologically reviewed and analyzed for periostin expression using immunohistochemistry. The intensities of immunoreactivity and the fraction of positive cancer cells and stroma (i.e., epithelial and stromal expression, respectively) were classified into four categories each (intensity, 0–3; fraction, 0–25% = 1; 26–50% = 2; 51–75% = 3; and > 75% = 4). The overall score was determined by multiplying both scores, and overall scores ≥ 6 were considered to indicate high periostin expression.ResultsAmong 126 UTUCs, 55 (44%; 27 renal pelvic and 28 ureteral cancers) showed high stromal periostin expression. None of the cases were considered to have high epithelial periostin expression. High stromal periostin expression was associated with non-papillary gross findings, higher pathological T category, lymphovascular invasion, concomitant carcinoma in situ, subtype histology, lymph node metastasis, positive surgical margins, high tumor budding, and high tumor-associated immune cell status. Multivariate analysis revealed that high stromal periostin expression was an independent predictor of overall survival (p = 0.00072, hazard ratio = 3.62), and lymphovascular invasion and high stromal periostin expression were independent predictors of cancer-specific survival (p = 0.032 and 0.020, hazard ratio = 2.61 and 3.07, respectively).ConclusionsStromal periostin expression was often observed in invasive UTUCs with adverse clinicopathological factors and may be a useful predictor of patient outcomes.

Deglycosylation of pathological specimens alters performance of diagnostic PDL1 antibodies.

Immunohistochemical (IHC) predictive quantitation of PDL1 expression is obligatory in many cancer entities with improved response to immune checkpoint inhibition in PDL1-positive subgroups. With recent demonstration of increased positivity rates after enzymatic deglycosylation in breast cancer specimens, a comparative analysis with two different antibodies and extended controls was performed in a cohort of head and neck squamous cell cancer samples (HNSCC).Formalin-fixed paraffin-embedded tissue from HNSCC specimens was used for initial on-slide method optimization based on the PNGase F assay. SDS-PAGE and immunoblotting with the PDL1 antibody 28-8 was performed to evaluate deglycosylation efficiency. A tissue micro array of n = 527 tissue cores of 181 patients with HNSCC was used to determine the effects of deglycosylation on staining pattern and intensity with PDL1 antibodies 28-8 and E1L3N.Successful on-slide deglycosylation with PNGase F was confirmed by immunoblot but varied across replicates. Using E1L3N (intracellular binding domain, most probably not glycosylated), mean signal intensity as well as the fraction of PDL1 positive cells was increased by deglycosylation. Opposite effects were observed with 28-8 (extracellular binding domain, glycosylated).Deglycosylation reduces diagnostic performance of the PDL1 antibody 28-8. In contrast, effects for E1L3N are complex and probably involve reduction of off-target binding leading to specifically improved signal intensity. However, enzymatic deglycosylation adds further variance to IHC.

The risk of PD-L1 expression misclassification in triple-negative breast cancer

PurposeStratification of patients with triple-negative breast cancer (TNBC) for anti-PD-L1 therapy is based on PD-L1 expression in tumor biopsies. This study sought to evaluate the risk of PD-L1 misclassification.MethodsWe conducted a high-resolution analysis on ten surgical specimens of TNBC. First, we determined PD-L1 expression pattern distribution via manual segmentation and measurement of 6666 microscopic clusters of positive PD-L1 immunohistochemical staining. Then, based on these results, we generated a computer model to calculate the effect of the positive PD-L1 fraction, aggregate size, and distribution of PD-L1 positive cells on the diagnostic accuracy.ResultsOur computer-based model showed that larger aggregates of PD-L1 positive cells and smaller biopsy size were associated with higher fraction of false results (P < 0.001, P < 0.001, respectively). Additionally, our model showed a significant increase in error rate when the fraction of PD-L1 expression was close to the cut-off (error rate of 12.1%, 0.84%, and 0.65% for PD-L1 positivity of 0.5–1.5%, ≤ 0.5% ,and ≥ 1.5%, respectively, P < 0.0001). Interestingly, false positive results were significantly higher than false negative results (0.51–22.62%, with an average of 6.31% versus 0.11–11.36% with an average of 1.58% for false positive and false negative results, respectively, P < 0.05). Furthermore, heterogeneous tumors with different aggregate sizes in the same tumor, were associated with increased rate of false results in comparison to homogenous tumors (P < 0.001).ConclusionOur model can be used to estimate the risk of PD-L1 misclassification in biopsies, with potential implications for treatment decisions.

Lymph Nodes Draining Infections Investigated by PET and Immunohistochemistry in a Juvenile Porcine Model.

Background: [18F]FDG and [11C]methionine accumulate in lymph nodes draining S. aureus -infected foci. The lymph nodes were characterized by weight, [11C]methionine- and [18F]FDG-positron emissions tomography (PET)/computed tomography (CT), and immunohistochemical (IHC)-staining. Methods: 20 pigs inoculated with S. aureus into the right femoral artery were PET/CT-scanned with [18F]FDG, and nine of the pigs were additionally scanned with [11C]methionine. Mammary, medial iliac, and popliteal lymph nodes from the left and right hind limbs were weighed. IHC-staining for calculations of area fractions of Ki-67, L1, and IL-8 positive cells was done in mammary and popliteal lymph nodes from the nine pigs. Results: The pigs developed one to six osteomyelitis foci. Some pigs developed contiguous infections of peri-osseous tissue and inoculation-site abscesses. Weights of mammary and medial iliac lymph nodes and their [18F]FDG maximum Standardized Uptake Values (SUVFDGmax) showed a significant increase in the inoculated limb compared to the left limb. Popliteal lymph node weight and their FDG uptake did not differ significantly between hind limbs. Area fractions of Ki-67 and IL-8 in the right mammary lymph nodes and SUVMetmax in the right popliteal lymph nodes were significantly increased compared with the left side. Conclusion: The PET-tracers [18F]FDG and [11C]methionine, and the IHC- markers Ki-67 and IL-8, but not L1, showed increased values in lymph nodes draining soft tissues infected with S. aureus. The increase in [11C]methionine may indicate a more acute lymph node response, whereas an increase in [18F]FDG may indicate a more chronic response.

Correlation and colocalization of HIF-1α and pimonidazole staining for hypoxia in laryngeal squamous cell carcinomas: A digital, single-cell-based analysis

ObjectiveTumor hypoxia results in worse local control and patient survival. We performed a digital, single-cell-based analysis to compare two biomarkers for hypoxia (hypoxia-inducible factor 1-alpha [HIF-1α] and pimonidazole [PIMO]) and their effect on outcome in laryngeal cancer patients treated with accelerated radiotherapy with or without carbogen breathing and nicotinamide (AR versus ARCON). Materials and MethodsImmunohistochemical staining was performed for HIF-1α and PIMO in consecutive sections of 44 laryngeal cancer patients randomized between AR and ARCON. HIF-1α expression and PIMO-binding were correlated using digital image analysis in QuPath. High-density areas for each biomarker were automatically annotated and staining overlap was analyzed. Kaplan-Meier survival analyses for local control, regional control and disease-free survival were performed to predict a response benefit of ARCON over AR alone for each biomarker. Results106 Tissue fragments of 44 patients were analyzed. A weak, significant positive correlation was observed between HIF-1α and PIMO positivity on fragment level, but not on patient level. A moderate strength correlation (r = 0.705, p < 0.001) was observed between the number of high-density staining areas for both biomarkers. Staining overlap was poor. HIF-1α expression, PIMO-binding or a combination could not predict a response benefit of ARCON over AR. ConclusionDigital image analysis to compare positive cell fractions and staining overlap between two hypoxia biomarkers using open-source software is feasible. Our results highlight that there are distinct differences between HIF-1α and PIMO as hypoxia biomarkers and therefore suggest co-existence of different forms of hypoxia within a single tumor.

Effects of Er:YAG laser treatment on re-vascularization and follicle survival in frozen/thawed human ovarian cortex transplanted to immunodeficient mice.

The huge loss of ovarian follicles after transplantation of frozen/thawed ovarian tissue is considered a major drawback on the efficacy of the procedure. Here we investigate whether Er:YAG laser treatment prior to xenotransplantation can improve re-vascularization and subsequently follicle survival in human ovarian tissue. A total of 99 frozen/thawed human ovarian cortex pieces were included of which 72 pieces from 12 woman were transplanted to immunodeficient mice. Tissues from each woman were included in both an 8-day and an 8-week duration study and treated with either full-beam laser (L1) or fractionated laser (L2), or served as untreated controls. Vascularization of the ovarian xenografts were evaluated after 8 days by qPCR and murine Cd31 immunohistochemical analysis. Follicle densities were evaluated histologically 8 weeks after xenografting. Gene expression of Vegf/VEGF was upregulated after L1 treatment (p=0.002, p=0.07, respectively), whereas Angpt1, Angpt2, Tnf-α, and Il1-β were significantly downregulated. No change in gene expression was found in Cd31/CD31, ANGPT1, ANGPT2, ANGTPL4, XBP1, or LRG1 after any of the laser treatments. The fraction of Cd31 positive cells were significantly reduced after L1 and L2 treatment (p<0.0001; p=0.0003, respectively), compared to controls. An overall negative effect of laser treatment was detected on follicle density (p=0.03). Er:YAG laser treatment did not improve re-vascularization or follicle survival in human ovarian xenografts after 8 days and 8 weeks grafting, respectively. However, further studies are needed to fully explore the potential angiogenic effects of controlled tissue damage using different intensities or lasers.

STRO-1 positive cell expansion during osteogenic differentiation: A comparative study of three mesenchymal stem cell types of dental origin

ObjectiveAlthough the osteogenic differentiation potential of mesenchymal stem cells of dental origin is well established, the roles of different marker proteins in this process remain to be clarified. Our aim was to compare the cellular and molecular changes, focusing in particular on mesenchymal stem cell markers, during in vitro osteogenesis in three dental stem cell types: dental follicle stem cells (DFSCs), periodontal ligament stem cells (PDLSCs) and dental pulp stem cells (DPSCs). DesignHuman DFSCs, PDLSCs and DPSCs were isolated, cultured and their osteogenic differentiation was induced for 3 weeks. Mineralization was assessed by von Kossa staining and calcium concentration measurements. The expression of mesenchymal and osteogenic markers was studied by immunocytochemistry and qPCR techniques. Alkaline phosphatase (ALP) activity and the frequency of STRO-1 positive cells were also quantified. ResultsThe three cultures all showed abundant mineralization, with high calcium content by day 21. The expression of vimentin and nestin was sustained after osteogenic induction. The osteogenic medium induced a considerable elevation of STRO-1 positive cells. By day 7, the ALP mRNA level had increased more than 100-fold in DFSCs, PDLSCs, and DPSCs. Quantitative PCR results indicated dissimilarities of osteoblastic marker levels in the three dental stem cell cultures. ConclusionsDFSCs, PDLSCs and DPSCs have similar functional osteogenic differentiation capacities although their expressional profiles of key osteogenic markers show considerable variations. The STRO-1 positive cell fraction expands during osteogenic differentiation while vimentin and nestin expression remain high. For identification of stemness, functional studies rather than marker expressions are needed.

Synergistic anticancer activity of resveratrol in combination with docetaxel in prostate carcinoma cells.

BACKGROUND/OBJECTIVESThe study was conducted to investigate the efficacy of the combination treatment of phytochemical resveratrol and the anticancer drug docetaxel (DTX) on prostate carcinoma LNCaP cells, including factors related to detailed cell death mechanisms.MATERIALS/METHODSUsing 2-dimensional monolayer and 3-dimensional spheroid culture systems, we examined the effects of resveratrol and DTX on cell viability, reactive oxygen species (ROS) levels, mitochondrial membrane potential, apoptosis, and necroptosis by MTT, flow cytometry, and Western blotting.RESULTSAt concentrations not toxic to normal human prostate epithelial cells, resveratrol effectively decreased the viability of LNCaP cells depending on concentration and time. The combination treatment of resveratrol and DTX exhibited synergistic inhibitory effects on cell growth, demonstrated by an increase in the sub-G0/G1 peak, Annexin V-phycoerythrin positive cell fraction, ROS, mitochondrial dysfunction, and DNA damage response as well as concurrent activation of apoptosis and necroptosis. Apoptosis and necroptosis were rescued by pretreatment with ROS scavenger N-acetylcysteine.CONCLUSIONSWe report resveratrol as an adjuvant drug candidate for improving the outcome of treatment in DTX therapy. Although the underlying mechanisms of necroptosis should be investigated comprehensively, targeting apoptosis and necroptosis simultaneously in the treatment of cancer can be a useful strategy for the development of promising drug candidates.

Abstract 358: Cancer cell derived TNF mRNA as a marker of cancer cell budding in colorectal cancer

Abstract Tumor necrosis factor alpha (TNF) is a potent cytokine reported to have several functions, including stimulation of inflammation, directing cell death and cell proliferation. The cellular origin of TNF has generally believed to be the inflammatory cells especially because macrophages are prone to induction of TNF. Using in situ hybridization (ISH) based on TNF mRNA probes (RNAscope) we recently reported that TNF mRNA is also seen in cytokeratin positive cancer cells (CC) and is associated with morphologic appearance interpreted as CC branching (Møller et al, IJMS 2019, doi: 10.3390/ijms20081907). To determine the fraction of CC-derived TNF mRNA signal at different areas in colon cancers we developed a cell and color segmentation-based image analysis tool to process fluorescence signals in digital slides. The approach allowed quantification of the TNF mRNA-positive CC and TNF mRNA-positive stromal cells in the digital slides previously reported, that were also stained for cytokeratin, microRNA-21 (miR-21) and DAPI nuclear stain. In the two cases with most abundant TNF mRNA ISH signal, both showing moderate-to-high budding index, the fraction of TNF-mRNA positive CC in the outer periphery (approximately 500 µm) was 8% and 20%, respectively. The frequency of the TNF-positive CC increased towards the invasive front from 7% (33/500 CC per 0.25 mm2) to 35% (48/137 CC), and from 8% (127/1536 CC per 0.50 mm2) to 28% (108/389 CC), respectively. The upregulation is in agreement with initial qualitative evaluation. In the same areas, the fraction of TNF mRNA positive stromal cells among all TNF mRNA-positive cells was only 8% in both cases, indicating that the majority of the TNF mRNA originates from CC. The fraction of the TNF mRNA-positive CC that were also miR-21 positive was 85% and 43%, respectively, which was higher than the prevalence of the overall miR-21 positive CC, 55% and 30%, suggesting frequent co-expression with miR-21. In conclusion, we have developed a method to obtain quantitative estimates from digital multiplex-stained slides with RNAscope and LNA fluorescence ISH and immunofluorescence. More samples will need to be processed to better understand the importance of TNF expression in cancer cell invasion. Citation Format: Boye Schnack Nielsen, Kim Holmstrom. Cancer cell derived TNF mRNA as a marker of cancer cell budding in colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 358.

Spatial heterogeneity of PD-L1 expression and the risk for misclassification of PD-L1 immunohistochemistry in non-small cell lung cancer

BackgroundIntra-tumor heterogeneity for PD-L1 expression in non-small cell lung cancer (NSCLC) might lead to inaccurate stratification of patients to immunotherapy. The purpose of this research was to quantitate the effect of different factors on the risk of inaccurate diagnosis of PD-L1 expression. MethodsMATLAB software was used to model tumor with a different fraction, distribution and clustering of PD-L1 protein expression and their effect on false positive and negative diagnosis in subsets of the modeled tumor (representing biopsies). Additionally, we evaluated the agreement between PD-L1 status in random segments and whole slides of PD-L1 stained clinical NSCLC cases. ResultsOur computer-based model showed a significant increase in error rate when the fraction of PD-L1 positive cells was closer to the cut-off value (error rate of 33.33 %, 0.45 % and 0.74 % for PD-L1 positivity in 40−60%, ≤20 % and ≥80 % of tumor cells, respectively, P < 0.0001). In addition, biopsy size showed negative correlation with error rate (P < 0.0001) and larger clusters of PD-L1 positive cells were associated with higher error rate (P < 0.0001). Analysis of the clinical samples supported those of the computer-based model with higher error rate in cases with positive PD-L1 expression closer to the cutoff value. Based on our computerized model and clinical analysis, we developed a model to predict error rate based on biopsy size and the fraction of PD-L1 positive cells in the biopsy. ConclusionAnalysis of small biopsies for PD-L1 expression might be associated with significant error rate. The model presented can be used to identify cases with increased risk for error in whom interpretation of the test results should be made with caution.

Investigating PSMA-Targeted Radioligand Therapy Efficacy as a Function of Cellular PSMA Levels and Intratumoral PSMA Heterogeneity.

Prostate-specific membrane antigen (PSMA) targeting radioligands deliver radiation to PSMA-expressing cells. However, the relationship between PSMA levels and intralesion heterogeneity of PSMA expression, and cytotoxic radiation by radioligand therapy (RLT) is unknown. Here we investigate RLT efficacy as function of PSMA levels/cell, and the fraction of PSMA+ cells in a tumor. RM1 cells expressing different levels of PSMA (PSMA-, PSMA+, PSMA++, PSMA+++; study 1) or a mix of PSMA+ and PSMA- RM1 (study 2, 4) or PC-3/PC-3-PIP (study 3) cells at various ratios were injected into mice. Mice received 177Lu- (studies 1-3) or 225Ac- (study 4) PSMA617. Tumor growth was monitored. Two days post-RLT, tumors were resected in a subset of mice. Radioligand uptake and DNA damage were quantified. 177Lu-PSMA617 efficacy increased with increasing PSMA levels (study 1) and fractions of PSMA positive cells (studies 2, 3) in both, the RM1 and PC-3-PIP models. In tumors resected 2 days post-RLT, PSMA expression correlated with 177Lu-PSMA617 uptake and the degree of DNA damage. Compared with 177Lu-PSMA617, 225Ac-PSMA617 improved overall antitumor effectiveness and tended to enhance the differences in therapeutic efficacy between experimental groups. In the current models, both the degree of PSMA expression and the fraction of PSMA+ cells correlate with 177Lu-/225Ac-PSMA617 tumor uptake and DNA damage, and thus, RLT efficacy. Low or heterogeneous PSMA expression represents a resistance mechanism to RLT.See related commentary by Ravi Kumar and Hofman, p. 2774.

Immunohistochemical detectability of cyclooxygenase-2 expression in cells of human melanocytic skin lesions: A methodological review.

Increased cyclooxygenase-2 (COX-2) expression is thought to support tumorigenesis through various mechanisms and is analyzed as a potential cancer marker. In 18 studies, COX-2 expression in melanocytic lesions of human skin was examined immunohistochemically. However, results obtained by individual research groups differ in terms of detection frequency and level of this protein, as well as localization of stained cells within tumor. Possible reasons for the discrepancies are analyzed in this review: the application of different antibodies, the use of standard histopathological sections or tissue microarrays and the analyzes of staining results based on different algorithms. COX-2 level is significantly lower in nevi than in melanomas, increases gradually with progression of these malignant cancers and reaches the highest values in metastases. These gradual changes in COX-2 expression appear to be difficult to analyze based only on subjective assessment of staining intensity. The most convergent data were obtained using antibodies for N-terminal fragments of COX-2 protein and analyzing results based on calculation of percentage fraction of positive cells. The extent of stained area in specimen thus appears to be more important than the intensity of staining in terms of evaluation of COX-2 performance as a diagnostic and prognostic marker of cutaneous melanoma.

P5385Development and preclinical testing of upscaled engineered heart tissue for use in translational studies

Abstract Introduction The lack of efficacy of stem cell therapy for the treatment of heart failure may be related to the poor retention rates offered by existing delivery methods (intra-coronary/ intramyocardial). Tissue engineering strategies improve cell retention in small animal models but data regarding engineered heart tissue (EHT) patches large enough for human studies are lacking. Purpose To upscale EHT to a clinically relevant size and mature the patch in-vitro. Once matured to undergo preclinical testing in a rabbit model of myocardial infarction. Methods We developed an upscaled EHT patch (3cm x 2cm x 1.5mm) able to contain up to 50 million human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CM; Fig A/B). Myocardial infarction model was performed by permanent ligation. Results The patches began to beat spontaneously within 3 days of fabrication and after 28 days of dynamic culture (Late EHTs) showed the development of several mature characteristics when compared to early patches (&lt;14 days from fabrication). For example, late EHTs contained hiPSC-CMs which were more aligned (hiPSC-CM accumulative angle change: early 2702±778 degrees [n=4] vs late 922±186 [n=5], p=0.042); showed better contraction kinetics (early peak contraction amplitude 87.9±5.8a.u. versus late 952±304a.u.; p&lt;0.001) and faster calcium transients (time to peak: early 200.8±8.8ms [n=5] vs late 147.7±10.2ms [n=6], p=0.004; time to 75% decay: early 274±9.7ms vs late 219.9±2.7ms, p=0.0003). We then tested the EHT patch in-vivo using a rabbit model (Fig C). Patches were applied to normal (n=5) or infarcted hearts (n=8). Sham operations used non-cellular fibrin patches (n=5). The mean fraction of troponin positive cells in the graft was 27.8±10.3% at 25.2±1.7 days relative to day 0 [n=5] and KU80 (human specific marker) staining confirmed that this was of human origin. CD31 (Fig D) and KU80 staining revealed that the grafts were well vascularized and that the vasculature was not human in origin (therefore were originating from the host). Ex-vivo optical mapping revealed evidence of electrical coupling between the graft and host at 2 weeks and preliminary experiments indicated that the patch improved left ventricular function when grafted onto infarcted hearts. Telemetry recordings in vivo and arrhythmia provocation protocols (ex vivo) indicated that the patch was not proarrhythmic. Figure 1. A/B) EHT Images; C) 20x troponin T (brown) of rabbit myocardium/EHT (2 weeks after grafting), blue counterstain = haematoxylin, red lines = EHT borders; D) 63x CD31 staining (brown) rabbit/EHT border zone (2 weeks after grafting), blue stain = haematoxylin, red lines = graft/host border zones. Conclusion We successfully upscaled hiPSC-CM derived EHT to a clinically relevant size and demonstrated feasibility and integration using a rabbit model of myocardial infarction. Tissue engineering strategies may be the preferred modality of cell delivery for future cardiac regenerative medicine studies.