Adherent Counterparts Research Articles

Treatment adherence and quality of life in colombian patients with lupus nephritis.

As with many other chronic diseases, systemic lupus erythematosus (SLE) and lupus nephritis (LN) have significant impacts on the health-related quality of life (HRQoL). Medication non-adherence is a significant challenge in the management of SLE, with consistently up to 75% of patients being non-adherent with their SLE medications. There is a need to assess the patient's perspective using patient-reported outcomes (PROs) to better understand the current impact of LN on HRQoL and treatment adherence in our region. The aim of this study was to explore the relationship between HRQoL and treatment adherence in patients with LN from the Colombian Caribbean. A cross-sectional study was conducted from June to December 2022, including patients with biopsy-proven LN. HRQoL and treatment adherence were assessed using the Lupus Quality of Life (LupusQoL) and the Compliance Questionnaire in Rheumatology 19 (CQR19) instruments, respectively. Patients were categorized as adherent or non-adherent based on medication intake (defined as >80% correct dosage). Principal component analysis (PCA) was employed to identify principal components between adherent and non-adherent patients. A total of 42 patients with LN were included. Of these, 38 (90%) were female, and the mean age was 31 ± 10 years. Proliferative class IV was the predominant histopathological profile (90%). Twenty-five (60%) patients were categorized as non-adherent. Across all LupusQoL domains, a comprehensive range of responses was observed. Pain, planning, and intimate relationships domains remained unaffected, while burden to others domain had the lowest score. Poorer planning score correlated with older age (r = -0.72; p < .05) and longer disease duration (r = -0.74; p < .05). SLEDAI-2K correlated with the pain domain (r = -0.78; p < .05). Non-adherent patients exhibited significantly worse pain domain scores compared to adherent counterparts (p < .05). PCA showed strong interactions between planning and pain, as well as between physical health and body image domains. LupusQoL pain domain scores were significantly worse in non-adherent patients compared to adherent patients. Effective pain management could be a determinant in HRQoL and treatment adherence rates in our population.

Human esophageal cancer stem-like cells escape the cytotoxicity of natural killer cells via down-regulation of ULBP-1

BackgroundCancer stem-like cells (CSCs) play an important role in initiation and progression of aggressive cancers, including esophageal cancer. Natural killer (NK) cells are key effector lymphocytes of innate immunity that directly attack a wide variety of cancer cells. NK cell-based therapy may provide a new treatment option for targeting CSCs. In this study, we aimed to investigate the sensitivity of human esophageal CSCs to NK cell-mediated cytotoxicity.MethodsCSCs were enriched from human esophageal squamous cell carcinoma cell lines via sphere formation culture. Human NK cells were selectively expanded from the peripheral blood of healthy donors. qRT-PCR, flow cytometry and ELISA assays were performed to examine RNA expression and protein levels, respectively. CFSE-labeled target cells were co-cultured with human activated NK cells to detect the cytotoxicity of NK cells by flow cytometry.ResultsWe observed that esophageal CSCs were more resistant to NK cell-mediated cytotoxicity compared with adherent counterparts. Consistently, esophageal CSCs showed down-regulated expression of ULBP-1, a ligand for NK cells stimulatory receptor NKG2D. Knockdown of ULBP-1 resulted in significant inhibition of NK cell cytotoxicity against esophageal CSCs, whereas ULBP-1 overexpression led to the opposite effect. Finally, the pro-differentiation agent all-trans retinoic acid was found to enhance the sensitivity of esophageal CSCs to NK cell cytotoxicity.ConclusionsThis study reveals that esophageal CSCs are more resistant to NK cells through down-regulation of ULBP-1 and provides a promising approach to promote the activity of NK cells targeting esophageal CSCs.

Gynecologic infection rates after ablation treatment for cervical intraepithelial neoplasia grade 2 and higher (CIN2+): Secondary analysis of a non-inferiority randomized trial.

Although concerns have been raised regarding potential infection and morbidity in women undergoing ablation treatment for cervical precancer in low- and middle-income countries (LMIC), there is extremely limited data to substantiate this claim. This is a secondary analysis of a randomized non-inferiority trial (id: NCT03084081) that compares the efficacy and safety of three ablation treatments for biopsy-confirmed cervical intraepithelial neoplasia grade 2 or higher (CIN2+): CO2 gas-based cryotherapy, non-gas cryotherapy, and thermal ablation (TA). Here, we present findings regarding the incidence of sexually transmitted infections (STI) and vaginitis post-treatment. Samples were collected at enrollment and again at 6 weeks post-treatment and assessed for STIs (Chlamydia trachomatis (CT), Neisseria gonorrhea (NG), and Trichomonas vaginalis (TV)) and vaginitis (Bacterial vaginosis (BV) and/or Candida albicans (Candida)). This analysis reflects 864 women with baseline and 6-week follow-up data. None of the ablative treatments put women at increased risk for STIs (CT, NG, TV) or vaginitis (BV, Candida). While most women adhered to post-treatment recommendations (97%) and no difference by treatment arm was observed, the incidence of STIs at follow-up in women that did not adhere with a given recommendation was higher compared to their adherent counterparts. The incidence of gynecologic infection did not increase with any of the ablation treatments from baseline to the six-week follow-up. Non-gas cryotherapy and TA emerge as safe alternatives to gas-based cryotherapy with respect to gynecologic infection rates.

PGC1-α-driven mitochondrial biogenesis contributes to a cancer stem cell phenotype in melanoma

Little is known about the metabolic regulation of cancer stem cells (CSCs) in melanoma. Here, we used A375 and WM115 cell lines to dissect the role of mitochondria in conferring CSC traits. Notably, we observed that A375 and WM115 melanospheres, known to be enriched in ABCG2+ CSCs, showed higher mitochondrial mass compared with their adherent counterpart. In particular, they displayed increased PGC1-α expression and oxidative phosphorylation (OXPHOS) complex levels, leading to a metabolic switch characterized by enhanced mitochondrial membrane potential, oxygen consumption, ATP synthesis and ROS production. Interestingly, PGC1-α silencing resulted in the suppression of CSC features, including clonogenic ability, migration, spheroid formation and ABCG2 enrichment. Similarly, XCT790 and SR-18292, two PGC1-α inhibitors, were able not only to reduce melanoma tumorigenicity and invasion but also to block melanosphere growth and propagation and ABCG2+ cell proliferation. In conclusion, improved mitochondrial biogenesis is associated with a stem-like phenotype in melanoma, and therapeutically targeting the mitochondria-enriched CSC subpopulation might overcome tumor progression.

BSBM-13 MELANOMA CELLS PRODUCE A SUSPENSION SUB-POPULATION THAT DRASTICALLY DISRUPTS THE BLOOD-BRAIN BARRIER ENDOTHELIUM

Abstract Melanoma metastasizes to the brain with high propensity. Brain metastasis requires cancer cells to breach the blood-brain barrier (BBB) endothelium, which forms the first line of defence against blood-borne pathogens. An increasing number of molecules are discussed in BBB disruption, and their importance varies across melanoma and endothelial cell lines. This diversity in literature proposes that cancer cells of the same cancer type employ different modes of metastasis. This study shows an example of the difference in migration capabilities of adherent melanoma cells and a suspension sub-population. We used Electric Cell-substrate Impedance Sensing (ECIS) to assess changes in human brain endothelial barrier function upon adding human melanoma lines. Several melanoma lines induced brain endothelial barrier disruption within the first few hours of addition. Intriguingly, the suspension population of melanoma cells disrupted the endothelial barrier faster and more drastically than the adherent counterpart. Time-lapse imaging revealed that the suspension population used a transcellular route of invasion compared to the paracellular route used by the adherent cells. Confocal microscopy supported this, showing clear differences in the physical interaction of the suspension subtype with the brain endothelium, compared to the adherent cells. Flow cytometry and NanoString technology allowed characterization of the melanoma lines, showing that the suspension subtype protein composition differs from their adherent counterpart. We hypothesize that melanoma cells may produce specific subtypes that are more invasive, which can accelerate migration through the brain endothelium. We stress the importance of investigating the composition of melanoma cells and their bi-products when researching cancer metastasis. Once successfully within the brain parenchyma, these cancer cells are effectively separated from the peripheral adaptive immune system, designed to detect and eliminate them. Therefore, preventing cancer cell migration into the brain is an attractive consideration, as they are considerably more targetable whilst still in the blood.

Association between statin use and fractures among dialysis patients in the north of the West Bank in 2021

IntroductionHip fractures are associated with higher morbidity and mortality among dialysis patients than among the general population. Statins reduce the risk of hip and bone fractures. However, this protective effect has not been studied in dialysis patients. MethodsHemodialysis patients from hemodialysis centers located in the north of the West Bank were recruited (N ​= ​713) in a cross-sectional study in 2021. A statin user is a patient who has used statin for at least one year. The patient's medical records and an interview-administered structured questionnaire were used. Adjusted multiple logistic regression examined the association between statin use and fractures. A p-value less than 0.05 was considered statistically significant. ResultsThe final sample size was 529 patients, 184 were excluded, 60.7% were males, 54.4% were statin users, 76.1% were always adherent to their statin, and 75.3% used moderate-intensity statin. Statin users who were always adherent to their dose had a significantly lower risk of hip fractures (OR ​= ​0.090, p-value ​= ​0.026) than rarely adherent patients. In addition, patients who were always adherent with their statin dose (OR ​= ​0.188, p-value ​= ​0.007) or sometimes adherent (OR ​= ​0.171, p-value ​= ​0.022) had a significantly lower risk of having other bone fractures after dialysis than the rarely adherent counterparts. There were no differences between high and moderate-intensity statins regarding hip fracture effects (p-value >0.05). Moreover, the increased duration of dialysis significantly increased the risk of hip fractures (OR ​= ​1.349, p-value ​= ​0.003). ConclusionThe results of this study suggest that adherence to statin therapy was associated with decreased risk of hip fractures and other bone fractures among hemodialysis patients. These results could be the initial evidence of the protective effect against a vital health hazard among dialysis patients. This might ultimately contribute to re-considering the current statins prescription practice for dialysis patients. Further studies are recommended.

Co-Treatment with Human Leukocyte Extract and Albendazole Stimulates Drug's Efficacy and Th1 Biased Immune Response in Mesocestoides vogae (Cestoda) Infection via Modulation of Transcription Factors, Macrophage Polarization, and Cytokine Profiles.

The model flatworm Mesocestoides vogae proliferating stage of infection elicits immunosuppression in the host. It was used to investigate the effects of human leukocyte extract (DLE) alone and in combination with anthelmintic albendazole (ABZ) on the reduction in peritoneal infection, peritoneal exudate cells (PECs), their adherent counterparts, and peritoneal exudates after the termination of therapy. Balb/c mice were infected with the larvae of M. vogae. PECs and adherent macrophages were studied via flow cytometry, mRNA transcript levels, and immunofluorescence. The cytokine levels were measured via ELISA and larvae were counted. ABZ significantly reduced larval counts (581.2 ± 65, p < 0.001), but the highest reduction was observed after combined treatment with ABZ and DLE (389.2 ± 119, p < 0.001) in comparison with the control. Compared to an infected group, the proportions of CD11b+CD19- myeloid cells with suppressive ability decreased after albendazole (ABZ) in combination with DLE, which was the most effective in the elevation of B cells and CD11b+F4/80mid/highMHCIIhigh macrophages/monocytes (22.2 ± 5.4%). Transcripts of the M2 macrophage markers (arginase 1, FIZZ-1, and Ym-1) were downregulated after DLE and combined therapy but not after ABZ, and the opposite trend was seen for iNOS. This contrasts with reduced ex vivo NO production by LPS-stimulated PECs from DLE and ABZ+DLE groups, where adherent macrophages/monocytes had elevated transcripts of the INF-γ receptor and STAT1 and reduced expression of STAT3, STAT6, and IL-10. Each therapy differentially modulated transcription profiles and concentrations of IFN-γ, TNF-α, IL-12p40, IL-6, IL-10, and TGF-β cytokines. DLE strongly ameliorated ABZ-induced suppression of INF-γ and IL-12 and preserved downregulation for IL-4, IL-10, and TGF-β. Epigenetic study on adherent macrophages from infected mice showed that ABZ, ABZ-sulfoxide, and DLE could interact with the mRNA of examined markers in a dose-dependent pattern. Co-administration of DLE with ABZ seemed to augment the drug's larvicidal effect via modulation of immunity. In comparison with ABZ, combined therapy was the most effective in alleviating parasite-induced Th2/Treg/STAT3/STA6 directed immunosuppression by stimulating the Th1 cytokines, M1 macrophage polarization, and activation of the IFNγ/STAT1 signaling pathway.

BCL6 and the Notch pathway: a signaling axis leading to a novel druggable biotarget in triple negative breast cancer.

The transcriptional repressor B-cell lymphoma 6 (BCL6) is dysregulated in several neoplasms, but its role in triple negative breast cancer (TNBC), a highly aggressive subtype which lacks effective treatment, is unclear. The presence of intratumoral cancer stem cells (CSCs) is a main cause of tumor relapse. The Notch signaling pathway is crucial for regulating CSC self-renewal and promoting breast cancer (BC) development and resistance to anticancer therapies. Here, we investigated signaling cascades of BCL6 in the CSC compartment of TNBCs, and the mechanisms that govern its activity, mainly through Notch signaling. Gene expression, somatic copy number alterations and clinical data from the Cancer Genome Atlas and METABRIC were accessed through theXena and cbioportal browsers. Public transcriptome profiles from TNBC datasets were retrieved from theGene Expression Omnibus. Mammosphere formation efficiency was calculated after BCL6 knockdown via transient siRNAtransfection, stable silencing or pharmacological inhibition. The effects exhibited via BCL6 inhibition in putative TNBC stem-like cells were evaluated by immunofluorescence and qRT-PCR analyses. Chromatin immunoprecipitation experiments were performed to validate a putative BCL6 responsive element located in the first intron of the Numb gene and to define the circuit of corepressors engaged by BCL6 following its inhibition. Immunoprecipitation assays were carried out to investigate a novel interaction at the basis of BCL6 control of CSC activity in TNBC. In silico analyses of benchmarked public datasets revealed a significant enrichment of BCL6 in cancer stemness related pathways, particularly of Notch signaling in TNBC. In vitro stable inhibition of BCL6 significantly reduced tumor cell growth and, accordingly, we found thatthe mammosphere formation efficiency of BCL6 silencedcells was significantly impaired by pharmacological inhibition of Notch signaling. BCL6 was found to be expressed at significantly higher levels in TNBC mammospheres than in their adherent counterparts, and loss of BCL6 function significantly decreased mammosphere formation with preferential targeting of CD44-positive versus ALDH-positive stem-like cells. Functional interplay between BCL6 and the chromatin remodeling factor EZH2 triggered the BCL6/Notch stemness signaling axis via inhibition of Numb transcription. Our results may be instrumental for the prospective design of combination treatment strategies that selectively target novel TNBC-associated biomarker(s) whose activity is implicated in the regulation of cancer stemness (such as BCL6) and molecules in developmentally conserved signaling pathways (such as Notch) to achieve long-lasting tumor control and improve patient outcomes.

Can ECIS Biosensor Technology Be Used to Measure the Cellular Responses of Glioblastoma Stem Cells?

Glioblastoma is considered the most aggressive and lethal form of brain cancer. Glioblastoma tumours are complex, comprising a spectrum of oncogenically transformed cells displaying distinct phenotypes. These can be generated in culture and are called differentiated-glioblastoma cells and glioblastoma stem cells. These cells are phenotypically and functionally distinct, where the stem-like glioblastoma cells give rise to and perpetuate the tumour. Electric cell-substrate impedance sensing (ECIS) is a real-time, label-free, impedance-based method for the analysis of cellular behaviour, based on cellular adhesion. Therefore, we asked the question of whether ECIS was suitable for, and capable of measuring the adhesion of glioblastoma cells. The goal was to identify whether ECIS was capable of measuring glioblastoma cell adhesion, with a particular focus on the glioblastoma stem cells. We reveal that ECIS reliably measures adhesion of the differentiated glioblastoma cells on various array types. We also demonstrate the ability of ECIS to measure the migratory behaviour of differentiated glioblastoma cells onto ECIS electrodes post-ablation. Although the glioblastoma stem cells are adherent, ECIS is substantially less capable at reliably measuring their adhesion, compared with the differentiated counterparts. This means that ECIS has applicability for some glioblastoma cultures but much less utility for weakly adherent stem cell counterparts.

Mammospheres of letrozole-resistant breast cancer cells enhance breast cancer aggressiveness.

Aromatase inhibitors (AIs), such as letrozole, are considered as first-line treatment for estrogen receptor-positive breast cancer in postmenopausal women. Despite the successful use of letrozole, resistance to therapy, tumor relapse and metastasis remain principal causes of patient mortality. Although there is no therapy currently available for AI-resistant breast cancer, previous reports have demonstrated that AI resistance is associated with hormone independence, increased growth factor signaling, enhanced cellular motility and epithelial to mesenchymal transition (EMT). This suggests a convergence of EMT and cancer stem cells (CSCs) in endocrine resistance. The present study evaluated the contribution of mammospheres in letrozole-resistant breast cancer by characterizing mammospheres and their potential impact on cellular motility. Ovariectomized immunocompromised female mice were inoculated in the mammary fat pad with either letrozole-resistant MCF-7 cells (LTLT-Ca) or letrozole-sensitive MCF-7 cells (AC-1). Subsequently, intratumoral CSC marker expression was assessed by immunohistochemistry. The results indicated that LTLT-Ca tumors were CD44+/CD24+, while AC-1 tumors presented low CD44/CD24 expression. Since mammosphere formation depends on CSCs, both cell lines were cultured either adherently (2D) or as mammospheres (3D) to assess the CD44/CD24 protein expression profile. When 3D culturing both cell lines, higher expression levels of CD44 and CD24 were observed when compared with their adherent counterparts, with the most robust change observed in the LTLT-Ca cell line. To quantitate the breast cancer stem cell activity, mammosphere formation assays were performed, and the LTLT-Ca cells formed mammospheres at a 3.4-fold higher index compared with AC-1 cells. Additionally, targeted gene expression arrays were conducted to compare the LTLT-Ca 3D and 2D cells, revealing that LTLT-Ca 3D cells displayed decreased expression levels of genes involved in cell adhesion and tumor suppression (e. g., E-cadherin, caveolin 1 and β-catenin). To validate this finding, wound healing assays were performed, and LTLT-Ca mammospheres exhibited a 70% wound closure, whereas AC-1 mammospheres exhibited a 39% wound closure. Collectively, the present findings demonstrated a strong association between AI-resistant mammospheres and an increased propensity for migration, which may be indicative of a poor prognosis.

A three-dimensional microenvironment alters CD73 expression in cervical cancer.

Stem-like cells (CSCs) have a tumour-initiating capacity and play critical role in tumour metastasis, relapse and resistance to therapy. The ectoenzyme CD73, encoded by the NT5E gene, which catalyses the hydrolysis of AMP into adenosine, has been associated to an immunosuppressive tumour microenvironment, tumour cell adhesion and migration. Therefore, we investigated the expression and activity of CD73 in sphere-forming cells from cervical cancer in comparison to monolayer cells in vitro. In addition, in silico analysis was performed to determine the expression of CD73 and other members of purinergic signalling in CSC-like population derived from different tumour types in comparison to monolayer cells. CD73 protein expression levels and functionality in SiHa cells were analysed by flow cytometry and enzymatic assay, respectively. In silico investigation was performed through the analysis of seven datasets from different tumour types using GEO database. In vitro analysis showed a decreased CD73 protein expression and enzymatic activity in cervical spheres, when compared to monolayers. In addition, when sphere-derived cells are re-plated as monolayer culture, the CD73 expression and activity are restored. Supporting the in vitro results, in silico analysis showed that three-dimensional spheres derived from cervical, thyroid and breast cancer presented decreased expression of CD73, when compared to their adherent counterparts. The decreased expression of CD73 in sphere-derived cells or CSC-enriched population reinforce its important role in cell adhesion, tumour spreading ability and metastasis, suggesting CD73 as potential target to be further investigated in cervical cancer.

Morphological and molecular characteristics of spheroid formation in HT-29 and Caco-2 colorectal cancer cell lines

BackgroundRelapse and metastasis in colorectal cancer (CRC) are often attributed to cancer stem-like cells (CSCs), as small sub-population of tumor cells with ability of drug resistance. Accordingly, development of appropriate models to investigate CSCs biology and establishment of effective therapeutic strategies is warranted. Hence, we aimed to assess the capability of two widely used and important colorectal cancer cell lines, HT-29 and Caco-2, in generating spheroids and their detailed morphological and molecular characteristics.MethodsCRC spheroids were developed using hanging drop and forced floating in serum-free and non-attachment conditions and their morphological features were evaluated by scanning electron microscopy (SEM). Then, the potential of CSCs enrichment in spheroids was compared to their adherent counterparts by analysis of serial sphere formation capacity, real-time PCR of key stemness genes (KLF4, OCT4, SOX2, NANOG, C-MYC) and the expression of potential CRC-CSCs surface markers (CD166, CD44, and CD133) by flow cytometry. Finally, the expression level of some EMT-related (Vimentin, SNAIL1, TWIST1, N-cadherin, E-cadherin, ZEB1) and multi-drug resistant (ABCB1, ABCC1, ABCG2) genes was evaluated.ResultsAlthough with different morphological features, both cell lines were formed CSCs-enriched spheroids, indicated by ability to serial sphere formation, significant up-regulation of stemness genes, SOX2, C-MYC, NANOG and OCT4 in HT-29 and SOX2, C-MYC and KLF4 in Caco-2 spheroids (p-value < 0.05) and increased expression of CRC-CSC markers compared to parental cells (p-value < 0.05). Additionally, HT-29 spheroids exhibited a significant higher expression of both ABCB1 and ABCG2 (p-value = 0.02). The significant up-regulation of promoting EMT genes, ZEB1, TWIST1, E-cadherin and SNAIL1 in HT-29 spheroids (p-value = 0.03), SNAIL1 and Vimentin in Caco-2 spheroids (p-value < 0.05) and N-cadherin down-regulation in both spheroids were observed.ConclusionEnrichment of CSC-related features in HT-29 and Caco-2 (for the first time without applying special scaffold/biochemical) spheroids, suggests spheroid culture as robust, reproducible, simple and cost-effective model to imitate the complexity of in vivo tumors including self-renewal, drug resistance and invasion for in vitro research of CRC-CSCs.

Quantitative Proteomic Profiling Identifies a Potential Novel Chaperone Marker in Resistant Breast Cancer.

Development of aromatase inhibitor resistant breast cancer among postmenopausal women continues to be a major clinical obstacle. Previously, our group demonstrated that as breast cancer cells transition from hormone-dependent to hormone-independent, they are associated with increased growth factor signaling, enhanced cellular motility, and the epithelial to mesenchymal transition (EMT). Given the complexity of cancer stem cells (CSC) and their implications on endocrine resistance and EMT, we sought to understand their contribution towards the development of aromatase inhibitor resistant breast cancer. Cells cultured three dimensionally as mammospheres are enriched for CSCs and more accurately recapitulates tumors in vivo. Therefore, a global proteomic analysis was conducted using letrozole resistant breast cancer cells (LTLT-Ca) mammospheres and compared to their adherent counterparts. Results demonstrated over 1000 proteins with quantitative abundance ratios were identified. Among the quantified proteins, 359 were significantly altered (p < 0.05), where 173 were upregulated and 186 downregulated (p < 0.05, fold change >1.20). Notably, midasin, a chaperone protein required for maturation and nuclear export of the pre-60S ribosome was increased 35-fold. Protein expression analyses confirmed midasin is ubiquitously expressed in normal tissue but is overexpressed in lobular and ductal breast carcinoma tissue as well as ER+ and ER- breast cancer cell lines. Functional enrichment analyses indicated that 19 gene ontology terms and one KEGG pathway were over-represented by the down-regulated proteins and both were associated with protein synthesis. Increased midasin was strongly correlated with decreased relapse free survival in hormone independent breast cancer. For the first time, we characterized the global proteomic signature of CSC-enriched letrozole-resistant cells associated with protein synthesis, which may implicate a role for midasin in endocrine resistance.

Screening for Diabetic Retinopathy with Extended Intervals, Safe and Without Compromising Adherence: A Retrospective Cohort Study.

IntroductionScreening for diabetic retinopathy (DR) prevents blindness through the early detection of sight-threatening retinal microvascular lesions that respond to timely local treatment. However, the provision of easy and regular access to DR screening programs is currently being challenged by the increasing prevalence of diabetes. One proposed solution is to extend the screening interval for patients at low risk for progression of retinopathy. To date, most providers of screening programs have hesitated to implement a strategy of extended intervals due to the lack of data on whether adherence and safety are compromised when retinal examinations occur less frequently. In the study reported here, we investigated adherence to the screening program and progression of retinopathy in patients with type 2 diabetes participating in a DR screening program with extended intervals.MethodsThis was a retrospective study that included 1000 patients with type 2 diabetes mellitus who attended a screening program for DR. The patients were consecutively placed into a low-risk patient cohort with no retinopathy or into an intermediate-risk patient cohort with mild retinopathy (each cohort n = 500). Screening intervals were 36 months for the low-risk cohort and 18 months for the intermediate-risk cohort.ResultsThe 1000 subjects enrolled in the study had a median age of 68 (interquartile range 12) years and 60.4% were men. At the follow-up screening visit, data on 102 subjects were not included in the analysis of adherence rate due to death, severe systemic illness, other concurrent eye disease or migration. Among the 898 remaining subjects, adherence to the screening program was 93.7% (413/443) in the 36-month group and 98.3% (449/455) in the 18-month group (p < 0.0001). Non-adherence decreased with increasing age (odds ratio 0.92, 95% confidence interval 0.888–0.954, p = 0.0005). At follow-up, 65 subjects showed progression of retinopathy; none had worse than moderate retinopathy. Risk factors for DR and treatment for hyperglycemia, hypertension and hyperlipidemia were compared among subjects in the low-risk cohort: non-adherent subjects did not differ from their adherent counterparts without progression of DR, but the former had a shorter duration of diabetes and higher diastolic blood pressure than adherent subjects with progression of DR (4.5 vs. 7.5 years, p = 0.007; and 80 vs. 75 mmHg, p = 0.02, respectively).ConclusionThe results suggest that screening DR at extended intervals can be achieved with high adherence rates without compromising patient safety. However, younger subjects and those at higher risk of progression may require extra attention.

CXCR4 Inhibition Counteracts Immunosuppressive Properties of Metastatic NSCLC Stem Cells.

Cancer stem cells (CSCs) are functionally defined as the cell subset with greater potential to initiate and propagate tumors. Within the heterogeneous population of lung CSCs, we previously identified highly disseminating CD133+CXCR4+ cells able to initiate distant metastasis (metastasis initiating cells-MICs) and to resist conventional chemotherapy. The establishment of an immunosuppressive microenvironment by tumor cells is crucial to sustain and foster metastasis formation, and CSCs deeply interfere with immune responses against tumors. How lung MICs can elude and educate immune cells surveillance to efficiently complete the metastasis cascade is, however, currently unknown. We show here in primary tumors from non-small cell lung cancer (NSCLC) patients that MICs express higher levels of immunoregulatory molecules compared to tumor bulk, namely PD-L1 and CD73, an ectoenzyme that catalyzes the production of immunosuppressive adenosine, suggesting an enhanced ability of MICs to escape immune responses. To investigate in vitro the immunosuppressive ability of MICs, we derived lung spheroids from cultures of adherent lung cancer cell lines, showing enrichment in CD133+CXCR4+MICs, and increased expression of CD73 and CD38, an enzyme that also concurs in adenosine production. MICs-enriched spheroids release high levels of adenosine and express the immunosuppressive cytokine IL-10, undetectable in an adherent cell counterpart. To prevent dissemination of MICs, we tested peptide R, a novel CXCR4 inhibitor that effectively controls in vitro lung tumor cell migration/invasion. Notably, we observed a decreased expression of CD73, CD38, and IL-10 following CXCR4 inhibition. We also functionally proved that conditioned medium from MICs-enriched spheroids compared to adherent cells has an enhanced ability to suppress CD8+ T cell activity, increase Treg population, and induce the polarization of tumor-associated macrophages (TAMs), which participate in suppression of T cells. Treatment of spheroids with anti-CXCR4 rescued T cell cytotoxic activity and prevented TAM polarization, likely by causing the decrease of adenosine and IL-10 production. Overall, we provide evidence that the subset of lung MICs shows high potential to escape immune control and that inhibition of CXCR4 can impair both MICs dissemination and their immunosuppressive activity, therefore potentially providing a novel therapeutic target in combination therapies to improve efficacy of NSCLC treatment.

Abstract 3794: Mammary cancer stem cells promote an aggressive phenotype in letrozole resistant breast cancer

Abstract Aromatase inhibitors (AI), like letrozole, are the first-line treatment for ER+ breast cancer in post-menopausal women. Despite widespread successful usage of letrozole, resistance to therapy, tumor relapse, and metastasis remain the principal causes of death for breast cancer patients. While there are no cures for AI-resistant breast cancer, previous reports demonstrate AI resistance is associated with hormone independence, increased growth factor signaling, enhanced cellular motility and the epithelial to mesenchymal transition (EMT). Given recent evidence suggesting a convergence of EMT and cancer stem cells (CSC) and their combined implications in endocrine resistance, we hypothesized that breast cancer stem cells contribute to letrozole resistant breast cancer cells by increasing cellular motility. To test this hypothesis, ovariectomized immunocompromised female mice were inoculated in the mammary fat pad with either MCF-7 cells stably transfected with the human aromatase gene that were long term letrozole-treated (LTLT-Ca) or letrozole sensitive, MCF-7 cells stably transfected with the human aromatase gene (AC-1). Afterwards, intratumoral putative CSC marker expression was assessed by immunohistochemistry. Results indicate LTLT-Ca tumors were CD44+/CD24+ while AC-1 tumors were CD44low/CD24low. Both cell lines were cultured either adherently (2D) or as mammospheres (3D) and CD44/CD24 protein expression was assessed by immunoblots. When both cell lines were cultured three dimensionally, they expressed higher levels of CD44 and CD24 compared to their adherent counterparts, with the most robust change occurring within the LTLT-Ca cells. In order to quantitate the breast cancer stem cell activity mammosphere formation assay was performed and LTLT-Ca cells formed mammospheres at a 3.4-fold higher index compared to AC-1 cells. Additionally, we performed targeted gene expression arrays and our results demonstrated decreased expression of genes involved in cell adhesion and tumor suppression (ie., E-cadherin, caveolin 1 and β catenin). Taken together our study demonstrated a strong correlation between AI resistance, increased cancer stem cell expression, an invasive phenotype that may be associated with a poor prognosis. This work was supported by an NIH grant awarded to SL Tilghman (1SC1GM125617). Citation Format: Karen Melissa Gallegos, Jankiben Patel, Rashidra R. Walker, A Michael Davidson, Ian R. Davenport, Syreeta L. Tilghman. Mammary cancer stem cells promote an aggressive phenotype in letrozole resistant breast 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 3794.

Spheroid-Derived Cells From Renal Adenocarcinoma Have Low Telomerase Activity and High Stem-Like and Invasive Characteristics.

Cancer stem cells (CSCs) are a theorized small subpopulation of cells within tumors thought to be responsible for metastasis, tumor development, disease progression, treatment-resistance, and recurrence. The identification, isolation, and biological characterization of CSCs may therefore facilitate the development of efficient therapeutic strategies targeting CSCs. This study aims to compare the biology and telomerase activity of CSCs to parental cells (PCs) in renal cancer. Renal CSCs were enriched from the ACHN cell line using a sphere culture system. Spheroid-derived cells (SDCs) and their adherent counterparts were compared with respect to their colony and sphere formation, expression of putative CSC markers, tumorigenicity in non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice, and invasiveness. The expression of genes associated with CSCs, stemness, EMT, apoptosis, and ABC transporters was also compared between the two populations using quantitative real-time PCR (qRT-PCR). Finally, telomerase activity, hTERT expression, and sensitivity to MST-312, a telomerase inhibitor, was investigated between the two populations. We demonstrated that a subpopulation of ACHN cells was capable of growing as spheroids with many properties similar to CSCs, including higher clonogenicity, superior colony- and sphere-forming ability, and stronger tumorigenicity and invasiveness. In addition, SDCs demonstrated a higher expression of markers for CSCs, stemness, EMT, apoptosis, and ABC transporter genes compared to PCs. The expression of hTERT and telomerase activity in SDCs was significantly lower than PCs; however, the SDC population was more sensitive to MST-312 compared to PCs. These findings indicate that the SDC population exhibits stem-like potential and invasive characteristics. Moreover, the reduced expression of hTERT and telomerase activity in SDCs demonstrated that the expressions of hTERT and telomerase activity are not always higher in CSCs. Our results also showed that MST-312 treatment inhibited SDCs more strongly than PCs and may therefore be useful as a complementary targeted therapy against renal CSCs in the future.

Nutritional and Metabolic Characteristics of UK Adult Phenylketonuria Patients with Varying Dietary Adherence.

The nutritional and metabolic characteristics of adult phenylketonuria (PKU) patients in the UK with varying dietary adherence is unknown. In other countries, nutritional and metabolic abnormalities have been reported in nonadherent patients compared to adherent counterparts. A pooled analysis of primary baseline data from two UK multi-centre studies was therefore performed to establish whether this is true from a UK perspective. Adult PKU patients who had provided 3-day food records and amino acid blood samples were included and grouped according to dietary adherence (adherent; n = 16 vs. nonadherent; n = 14). Nonadherent patients consumed greater amounts of natural protein compared to adherent patients (61.6 ± 30.7 vs. 18.3 ± 7.7 g/day; q < 0.001). In contrast, the contribution of protein substitutes to total protein intake was lower in nonadherent compared to adherent patients (3.9 ± 9.2 g/day vs. 58.6 ± 10.2 g/day; q < 0.001). Intakes of iron, zinc, vitamin D3, magnesium, calcium, selenium, iodine, vitamin C, vitamin A and copper were significantly lower in nonadherent compared to adherent patients and were below UK Reference Nutrient Intakes. Similarly, intakes of thiamin, riboflavin, niacin, vitamin B6 and phosphorus were significantly lower in nonadherent compared to adherent patients but met the UK Reference Nutrient Intakes. Phenylalanine concentrations in nonadherent patients were significantly higher than adherent patients (861 ± 348 vs. 464 ± 196 µmol/L; q = 0.040) and fell outside of European treatment target ranges. This study shows the nutritional and metabolic consequences of deviation from phenylalanine restriction and intake of PKU protein substitutes in nonadherent adult PKU patients. Collectively, these data further underlie the importance of life-long adherence to the PKU diet.

Weight loss and metabolic health effects from energy-restricted Mediterranean and Central-European diets in postmenopausal women: A randomized controlled trial

We conducted a randomized controlled trial to examine the effect of two energy-restricted diets on body weight (BW), visceral fat (VF) loss, and the risk factors for metabolic syndrome. A total of 144 centrally obese postmenopausal women were assigned to the moderate in fat Mediterranean diet (MED) or to the Central European diet (CED), which is moderate in carbohydrates and high in dietary fiber (DF), for 16 weeks. BW, waist circumference and VF were significantly reduced by 8.8%, 7.0%, and 24.6%, respectively, over the trial (P < 0.001), with no difference between groups. A similar trend was seen for total cholesterol, triglycerides, glucose, and blood pressure. Within each diet group, the more adherent participants lost significantly more BW than did their less adherent counterparts. VF was significantly reduced only in women who were more adherent to the CED, and the reduction in VF correlated with an increase in the proportion of DF. Short-term dietary treatment with the CED or the MED was associated with similar improvements in some anthropometric, lipid, and nonlipid parameters; however, adequate adherence to the prescribed diet is important in weight loss success and in achieving improvements in metabolic health.

Spheroids from adipose‐derived stem cells exhibit an miRNA profile of highly undifferentiated cells

Two-dimensional (2D) cell cultures have been extensively used to investigate stem cell biology, but new insights show that the 2D model may not properly represent the potential of the tissue of origin. Conversely, three-dimensional cultures exhibit protein expression patterns and intercellular junctions that are more representative of their in vivo condition. Multiclonal cells that grow in suspension are defined as "spheroids," and we have previously demonstrated that spheroids from adipose-derived stem cells (S-ASCs) displayed enhanced regenerative capability. With the current study, we further characterized S-ASCs to further understand the molecular mechanisms underlying their stemness properties. Recent studies have shown that microRNAs (miRNAs) are involved in many cellular mechanisms, including stemness maintenance and proliferation, and adipose stem cell differentiation. Most studies have been conducted to identify a specific miRNA profile on adherent adipose stem cells, although little is still known about S-ASCs. In this study, we investigate for the first time the miRNA expression pattern in S-ASCs compared to that of ASCs, demonstrating that cell lines cultured in suspension show a typical miRNA expression profile that is closer to the one reported in induced pluripotent stem cells. Moreover, we have analyzed miRNAs that are specifically involved in two distinct moments of each differentiation, namely early and late stages of osteogenic, adipogenic, and chondrogenic lineages during long-term in vitro culture. The data reported in the current study suggest that S-ASCs have superior stemness features than the ASCs and they represent the true upstream stem cell fraction present in adipose tissue, relegating their adherent counterparts.