Proliferative Potential Research Articles

Development of an alginate–chitosan biopolymer composite with dECM bioink additive for organ-on-a-chip articular cartilage

In vitro use of articular cartilage on an organ-on-a-chip (OOAC) via microfluidics is challenging owing to the dense extracellular matrix (ECM) composed of numerous protein moieties and few chondrocytes, which has limited proliferation potential and microscale translation. Hence, this study proposes a novel approach for using a combination of biopolymers and decellularised ECM (dECM) as a bioink additive in the development of scalable OOAC using a microfluidic platform. The bioink was tested with native chondrocytes and mesenchymal stem cell-induced chondrocytes using biopolymers of alginate and chitosan composite hydrogels. Two-dimensional (2D) and three-dimensional (3D) biomimetic tissue construction approaches have been used to characterise the morphology and cellular marker expression (by histology and confocal laser scanning microscopy), viability (cell viability dye using flow cytometry), and genotypic expression of ECM-specific markers (by quantitative PCR). The results demonstrated that the bioink had a significant impact on the increase in phenotypic and genotypic expression, with a statistical significance level of p < 0.05 according to Student’s t-test. The use of a cell-laden biopolymer as a bioink optimised the niche conditions for obtaining hyaline-type cartilage under culture conditions, paving the way for testing mechano-responsive properties and translating these findings to a cartilage-on-a-chip microfluidics system.

Modulation of Canine Adipose-Derived Mesenchymal Stem/Medicinal Signalling Cells with Ascorbic Acid: Effect on Proliferation and Chondrogenic Differentiation on Standard Plastic and Silk Fibroin Surfaces.

Ascorbic acid (AA) plays a crucial role in both the proliferation and chondrogenic differentiation potential of mesenchymal stem/medicinal signalling cells (MSCs); these are both key aspects of their general therapeutic use and their increasing use in veterinary medicine. Current immunomodulatory therapies require efficient expansion of MSCs in the laboratory, while emerging tissue regeneration strategies, such as cartilage or bone repair, aim to use differentiated MSCs and modulate the expression of chondrogenic and hypertrophic markers. Our aim was to investigate whether the addition of AA to the growth medium enhances the proliferation of canine adipose-derived MSCs (cAMSCs) grown on standard plastic surfaces and whether it affects chondrogenic differentiation potential on silk fibroin (SF) films. We assessed cell viability with trypan blue and proliferation potential by calculating population doubling. Chondrogenic induction on SF films was assessed by Alcian blue staining and gene expression analysis of chondrogenic and hypertrophic genes. The results showed that growth medium with AA significantly enhanced the proliferation of cAMSCs without affecting cell viability and modulated the expression of chondrogenic and hypertrophic genes of cAMSCs grown on SF films. Our results suggest that AA may be used in growth medium for expansion of cAMSCs and, at the same time, provide the basis for future studies to investigate the role of AA and SF in chondrogenic differentiation of MSCs.

Identification of the Wound Healing Activity Peptidome of Edible Bird's Nest Protein Hydrolysate and the In Silico Evaluation of Its Transport and Absorption Potential in Skin.

In this study, edible bird's nest (EBN) was proven to be a suitable source of bioactive peptides via enzymatic hydrolysis. The ultrafiltration component of the EBN peptides (EBNPs, Mw < 3 000 Da) could be responsible for moderate moisture retention and filaggrin synthesis. It was found that EBNP had a great capacity to protect HaCaT keratinocytes from DNA damage caused by UVB-irradiation and enhance wound healing by increasing the migratory and proliferative potential of cells. Furthermore, the external application of EBNP could effectively repair high glycolic acid concentration-induced skin burns in mice. A total of 1 188 peptides, predominantly the hydrophobic amino acids (e.g., Leu, Val, Tyr, Phe), were identified in the EBNP by liquid chromatography with tandem mass spectrometry (LC-MS/MS). Molecular docking showed that hydrophobic tripeptides from EBNP had a good binding affinity to proton-dependent oligopeptide transporter PepT1. Our data indicated that the hydrophobic amino acid-rich EBNP plays an important role in skin wound healing.

Comparative characteristics of the stem cells’ number in the stromal vascular fraction of infrapatellar fat pad and subcutaneous fat tissue

ObjectivesThe use of infrapatellar fat pad adipose stem cells (IPFP-ASCs) shows an age-independent proliferation and differentiation potential. In addition, the pronounced chondrogenic potential of IPFP-ASCs makes them promising candidates for research for use in other methods of regenerative therapy. The purpose of this study was to ascertain the presence and compare the relative abundance of cells exhibiting an immunohistochemical profile characteristic of adipose-derived mesenchymal stem cells in selected samples of the stromal vascular fraction (SVF) obtained from the IPFP and subcutaneous fat tissue. MethodsA direct immunohistochemical study was carried out in serial paraffin sections of the SVF of the infrapatellar fat pad (IPFP) and subcutaneous tissue, using monoclonal antibodies. The minimum criteria were established by the International Society for Cell Therapy to ensure the identity of mesenchymal stem cells use CD73, CD90, and CD105 as positive markers and CD34, CD31, and CD45 as a negative. ResultsAccording to the results of histological, immunohistochemical, morphometric, and statistical studies, it was found that in the SVF of IPFP and subcutaneous adipose tissue, the relative number of cells with the profile CD105+, CD73+, CD34+, CD31−, CD45− in the standard field of view (×200), the SVF of IPFP was 1.58%, whereas the SVF of subcutaneous adipose tissue was 6.92 %, which was statistically significantly greater by 4.38 times (p ​< ​0.05). ConclusionThe presence of a sufficient number of mesenchymal stromal cells in IPFP in combination with their topographic relationship with the structures of the joint determines the use of the SVF of the IPFP for the treatment of diseases of the knee joint. Level of evidenceIII.

Neutrophil-to-lymphocyte ratio in colorectal tissue affects prognosis in patients with colorectal cancer

The objective of this investigation was to analyse the correlation between the neutrophil-to-lymphocyte ratio (NLR) status in the immune microenvironment (IME) and the prognostic outcomes of patients who have undergone radical surgery for colorectal cancer (CRC). In light of the continued prevalence of CRC in China, this study utilised Kaplan-Meier and Cox regression analyses to assess the prognostic relevance of NLR status in IME among patients with CRC. Furthermore, cellular experiments, such as cell scratching, were conducted to elucidate the underlying mechanisms of NLR's impact on CRC. The NLR status in IME has been found to have a significant impact on the prognosis of patients with CRC. Patients who exhibit elevated intratumoural and extratumoural NLR are associated with a poor prognosis. Experimental evidence indicates that tumour-associated neutrophil (TAN) augments the migratory, invasive, and proliferative potential of HT-29, HCT-116 and LOVO colorectal cancer cells, while concurrently reducing their sensitivity to oxaliplatin. Conversely, lymphocytes have demonstrated cytotoxic effects on HT-29 cells. The NLR status in IME may serve as a prognostic biomarker for resectable CRC.

Carbon nanotubes conjugated with cisplatin activate different apoptosis signaling pathways in 2D and 3D-spheroid triple-negative breast cancer cell cultures: a comparative study

The type of experimental model for the in vitro testing of drug formulations efficiency represents an important tool in cancer biology, with great attention being granted to three-dimensional (3D) cultures as these offer a closer approximation of the clinical sensitivity of drugs. In this study, the effects induced by carboxyl-functionalized single-walled carbon nanotubes complexed with cisplatin (SWCNT–COOH–CDDP) and free components (SWCNT–COOH and CDDP) were compared between conventional 2D- and 3D-spheroid cultures of human breast cancer cells. The 2D and 3D breast cancer cultures were exposed to various doses of SWCNT–COOH (0.25–2 μg/mL), CDDP (0.158–1.26 μg/mL) and the same doses of SWNCT–COOH–CDDP complex for 24 and 48 h. The anti-tumor activity, including modulation of cell viability, oxidative stress, proliferation, apoptosis, and invasion potential, was explored by spectrophotometric and fluorometric methods, immunoblotting, optical and fluorescence microscopy. The SWCNT–COOH–CDDP complex proved to have high anti-cancer efficiency on 2D and 3D cultures by inhibiting cell proliferation and activating cell death. A dose of 0.632 μg/mL complex triggered different pathways of apoptosis in 2D and 3D cultures, by intrinsic, extrinsic, and reticulum endoplasmic pathways. Overall, the 2D cultures showed higher susceptibility to the action of complex compared to 3D cultures and SWCNT–COOH–CDDP proved enhanced anti-tumoral activity compared to free CDDP.

PPP2R2A inhibition contributes to preeclampsia by regulating the proliferation, apoptosis, and angiogenesis modulation potential of mesenchymal stem cells

BackgroundThe precise mechanisms underlying preeclampsia (PE) pathogenesis remain unclear. Mesenchymal stem cells (MSCs) are involved in the pathology of PE. The aim of our study was to identify the effects of protein phosphatase 2 regulatory subunit B α (PPP2R2A) on MSCs and ascertain its latent role in the progression of PE.MethodsReverse-transcription quantitative polymerase chain reaction and western blot analyses were performed to determine the expression of PPP2R2A in decidual tissue and decidual (d)MSCs from healthy pregnant women and patients with PE as well as the expression levels of Bax and Bcl-2 in dMSCs. The levels of p-PI3K, PI3K, p-AKT, and AKT were determined using western blotting. Cell growth, apoptosis, and migration were analyzed using MTT, flow cytometry, and Transwell assays, respectively. Human umbilical vein endothelial cell (HUVEC) tube formation ability was assayed using a HUVEC capillary-like tube formation assay.ResultsPPP2R2A was downregulated in decidual tissues and dMSCs of patients with PE when compared with that in healthy pregnant women. Moreover, upregulation of PPP2R2A enhanced cell proliferation, reduced apoptotic dMSC, inhibited Bax expression, and increased Bcl-2 levels. Conditioned medium from PPP2R2A-overexpressing dMSCs promoted HTR-8/SVneo cell migration and angiogenesis of HUVEC. Furthermore, the PPP2R2A plasmid suppressed PI3K/AKT pathway activation in dMSCs. However, these effects were partially reversed by LY2940002 treatment.ConclusionPPP2R2A inhibition contributes to PE by regulating the proliferation, apoptosis, and angiogenesis of MSCs, providing a new therapeutic target for PE diagnosis and treatment.

Cyclosporin A inhibits PDGF-BB induced hyaluronan synthesis in orbital fibroblasts

Orbital connective tissue changes are contributors to the pathogenesis in thyroid eye disease (TED). Activated fibroblasts respond to immune stimuli with proliferation and increased hyaluronan (HA) production. Cyclosporin A (CsA) was reported to be beneficial in the treatment of TED. PDGF isoforms are increased in orbital tissue of TED patients and enhance HA production. We aimed to study the effect of CsA on HA production and hyaluronan synthase (HAS1, 2 and 3) and hyaluronidase (HYAL1 and 2) mRNA expressions in orbital fibroblasts (OFs). Measurements were performed in the presence or absence of CsA (10 μM) in unstimulated or PDGF-BB (10 ng/ml) stimulated OFs. The HA production of TED OFs (n = 7) and NON-TED OFs (n = 6) were measured by ELISA. The levels of mRNA expressions were examined using RT-PCR. The proliferation rate and metabolic activity were measured by BrdU incorporation and MTT assays, respectively. Treatment with CsA resulted in an average 42% decrease in HA production of OFs (p < 0.0001). CsA decreased the expression levels of HAS2, HAS3 and HYAL2 (p = 0.005, p = 0.005 and p = 0.002, respectively.) PDGF-BB increased HA production (p < 0.001) and HAS2 expression (p = 0.004). CsA could reduce the PDGF-BB-stimulated HA production (p < 0.001) and HAS2 expression (p = 0.005) below the untreated level. In addition, CsA treatment caused a decrease in proliferation potential (p = 0.002) and metabolic activity (p < 0.0001). These findings point to the fact that CsA affects HA metabolism via HAS2, HAS3 and HYAL2 inhibition in OFs. In addition to its well characterized immunosuppressant properties, CsA's beneficial effect in TED may be related to its direct inhibitory effect on basal and growth factor stimulated HA production.

MerTK Drives Proliferation and Metastatic Potential in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is characterized by the absence of the estrogen receptor, progesterone receptor, and receptor tyrosine kinase HER2 expression. Due to the limited number of FDA-approved targeted therapies for TNBC, there is an ongoing need to understand the molecular underpinnings of TNBC for the development of novel combinatorial treatment strategies. This study evaluated the role of the MerTK receptor tyrosine kinase on proliferation and invasion/metastatic potential in TNBC. Immunohistochemical analysis demonstrated MerTK expression in 58% of patient-derived TNBC xenografts. The stable overexpression of MerTK in human TNBC cell lines induced an increase in proliferation rates, robust in vivo tumor growth, heightened migration/invasion potential, and enhanced lung metastases. NanoString nCounter analysis of MerTK-overexpressing SUM102 cells (SUM102-MerTK) revealed upregulation of several signaling pathways, which ultimately drive cell cycle progression, reduce apoptosis, and enhance cell survival. Proteomic profiling indicated increased endoglin (ENG) production in SUM102-MerTK clones, suggesting that MerTK creates a conducive environment for increased proliferative and metastatic activity via elevated ENG expression. To determine ENG's role in increasing proliferation and/or metastatic potential, we knocked out ENG in a SUM102-MerTK clone with CRISPR technology. Although this ENG knockout clone exhibited similar in vivo growth to the parental SUM102-MerTK clone, lung metastasis numbers were significantly decreased ~4-fold, indicating that MerTK enhances invasion and metastasis through ENG. Our data suggest that MerTK regulates a unique proliferative signature in TNBC, promoting robust tumor growth and increased metastatic potential through ENG upregulation. Targeting MerTK and ENG simultaneously may provide a novel therapeutic approach for TNBC patients.

The FAcilitates Chromatin Transcription complex regulates the ratio of glycolysis to oxidative phosphorylation in neural stem cells.

Defects in the FAcilitates Chromatin Transcription (FACT) complex, a histone chaperone composed of SSRP1 and SUPT16H, are implicated in intellectual disability. Here, we reveal that the FACT complex promotes glycolysis and sustains the correct cell fate of neural stem cells/neuroblasts in the Drosophila 3rd instar larval central brain. We show that the FACT complex binds to the promoter region of the estrogen-related receptor (ERR) gene and positively regulates ERR expression. ERR is known to act as an aerobic glycolytic switch by upregulating the enzymes required for glycolysis. Dysfunction of the FACT complex leads to the downregulation of ERR transcription, resulting in a decreased ratio of glycolysis to oxidative phosphorylation (G/O) in neuroblasts. Consequently, neuroblasts exhibit smaller cell sizes, lower proliferation potential, and altered cell fates. Overexpression of ERR or suppression of mitochondrial oxidative phosphorylation in neuroblasts increases the relative G/O ratio and rescues defective phenotypes caused by dysfunction of the FACT complex. Thus, the G/O ratio, mediated by the FACT complex, plays a crucial role in neuroblast cell fate maintenance. Our study may shed light on the mechanism by which mutations in the FACT complex lead to intellectual disability in humans.

Designing and synthesis of injectable hydrogel based on carboxymethyl cellulose/carboxymethyl chitosan containing QK peptide for femoral head osteonecrosis healing

Femoral head necrosis is a debilitating disorder that typically caused by impaired blood supply to the hip joint. In this study, a novel injectable hydrogel based on Oxidized Carboxymethyl Cellulose (OCMC)-Carboxymethyl Chitosan (CMCS) polymers containing an angiogenesis stimulator peptide (QK) with a non-toxic crosslinking interaction (Schiff based reaction) was synthesized to enhance angiogenesis following femoral head necrosis in an animal model. The physicochemical features of fabricated injectable hydrogel were analyzed by FTIR, swelling and degradation rate, rheometry, and peptide release. Also, the safety and efficacy were evaluated following an in vitro hydrogel injection study and an avascular necrosis (AVN) animal model. According to the results, the hydrogel exhibited an appropriate swelling ratio and water uptake (>90 %, 24 h) as well as a suitable degradation rate over 21 days accompanied by a continuous peptide release. Also, data showed that hydrogels containing QK peptide boosted the proliferation, differentiation, angiogenesis, and osteogenic potential of both Bone Marrow mesenchymal Stem Cells (BM-MSCs) and human umbilical vein endothelial cells (HUVECs) (****p < 0.0001 and ***p < 0.001, respectively). Furthermore, molecular and histological evaluations significantly demonstrated the overexpression of Runx2, Osteocalcin, Collagen I, VEGF and CD34 genes (**p < 0.01 and ***p < 0.001, respectively), and also femoral head necrosis was effectively prohibited, and more blood vessels were detected in defect area by OCMC-CMCS hydrogel containing QK peptide (bone trabeculae >9000, ***p < 0.001). In conclusion, the findings demonstrate that OCMC-CMCS-QK injectable hydrogel could be considered as an impressive therapeutic construct for femoral head AVN healing.

KiSS-1 Modulation by Epigenetic Agents Improves the Cisplatin Sensitivity of Lung Cancer Cells.

Epigenetic alterations my play a role in the aggressive behavior of Non-Small Cell Lung Cancer (NSCLC). Treatment with the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA, vorinostat) has been reported to interfere with the proliferative and invasive potential of NSCLC cells. In addition, the DNA methyltransferase inhibitor azacytidine (AZA, vidaza) can modulate the levels of the metastasis suppressor KiSS-1. Thus, since cisplatin is still clinically available for NSCLC therapy, the aim of this study was to evaluate drug combinations between cisplatin and SAHA as well as AZA using cisplatin-sensitive H460 and -resistant H460/Pt NSCLC cells in relation to KiSS-1 modulation. An analysis of drug interaction according to the Combination-Index values indicated a more marked synergistic effect when the exposure to SAHA or AZA preceded cisplatin treatment with respect to a simultaneous schedule. A modulation of proteins involved in apoptosis (p53, Bax) was found in both sensitive and resistant cells, and compared to the treatment with epigenetic agents alone, the combination of cisplatin and SAHA or AZA increased apoptosis induction. The epigenetic treatments, both as single agents and in combination, increased the release of KiSS-1. Finally, the exposure of cisplatin-sensitive and -resistant cells to the kisspeptin KP10 enhanced cisplatin induced cell death. The efficacy of the combination of SAHA and cisplatin was tested in vivo after subcutaneous inoculum of parental and resistant cells in immunodeficient mice. A significant tumor volume inhibition was found when mice bearing advanced tumors were treated with the combination of SAHA and cisplatin according to the best schedule identified in cellular studies. These results, together with the available literature, support that epigenetic drugs are amenable for the combination treatment of NSCLC, including patients bearing cisplatin-resistant tumors.

Dysregulation of the Long Noncoding RNA X-Inactive–Specific Transcript Expression in Male Patients with Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is a sex-biased disease with female gender as a significant risk factor. Recently, we reported that increased expression of the long non-coding (lnc)RNA Xist, as induced by an intersectin-1s protein fragment with proliferative potential (EHITSN), may explain the sexual dimorphism of female pulmonary artery endothelial cells (ECs) and at least in part, the imbalance sex/ratio of PAH.Xist is essential for X-chromosome inactivation and dosage compensation of X-linked genes. Increased Xist expression was also detected in a subset of ECs and lung tissue samples of male PAH patients. The role of different Xist expression levels in ECs of male PAH patients (ECPAH) was studied in several lines of male ECPAH in conjunction with molecular, biochemical, morphological, and functional approaches. Male ECPAH showed on average 10.3-fold increase in high Xist vs. low Xist, a significant association between Xist levels and their proliferative potential, and a heterogeneous methylation of the Xist/Tsix locus. Interestingly, Xist up-regulation in male ECPAH decreases the expression of Klf2, via EHITSN interaction with EZH2, the catalytic subunit of the polycomb repressive complex 2. Moreover, the studies demonstrate that EHITSN-triggered p38/Elk1/c-Fos signaling is a pathological mechanism central to ECPAH proliferation and the dynamic crosstalk with cell cycle regulatory proteins ccna1/ccnd2, and Xist-EZH2-Klf2 interaction participate directly and differentially in establishing the proliferative profile of male ECPAH.

Abstract PO2-04-14: Dual Inhibition of WEE1 and PKMYT1 Synergistically Overcomes CDK4/6 Inhibitor Resistance in Breast Cancer

Abstract Cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) are approved for the treatment of advanced estrogen receptor-positive, HER2-negative (ER+ve) breast cancer when combined with endocrine therapy. However, the emergence of resistance to CDK4/6 inhibitors poses a significant clinical challenge. This study investigates the mechanisms underlying CDK4/6 inhibitor resistance and identifies a common occurrence of high replication stress in triple-negative breast cancer (TNBC) and CDK4/6 inhibitor-resistant ER+ve breast cancers. While ER+ve breast cancer patients have benefited from targeted therapy, TNBC patients, particularly those without germline BRCA1/2 or PALB2 mutations, lack effective treatment options. TNBC and CDK4/6 inhibitor-resistant ER+ve breast cancer cells exhibit an increased dependence on cell cycle arrest to repair replication stress-induced DNA damage. Failure to exit the cell cycle results in excessive DNA damage, thereby limiting their proliferative potential. Based on these findings, we hypothesize that dual inhibition of WEE1 and PKMYT1, two kinases involved in G2/M cell cycle transition and crucial for cell cycle arrest and DNA damage repair, could synergistically inhibit tumor growth. Although WEE1 and PKMYT1 share a common function in promoting G2/M cell cycle transition through CDK1 phosphorylation, their functions are not entirely redundant, suggesting that targeting both kinases may elicit a synergistic response. Recent clinical trials have shown resistance to WEE1-targeted monotherapies, emphasizing the need for combining WEE1 inhibitors with other agents, such as PKMYT1 inhibitors. We demonstrate the synergistic effects of WEE1 inhibitor (MK1775/AZD1775) and PKMYT1 inhibitor (RP6306) in a panel of TNBC and ER+ve breast cancer cells with acquired palbociclib resistance (PalboR). Combination treatment with both inhibitors leads to excessive unrepaired DNA damage, as indicated by increased γH2AX foci following 48 hours of combination treatment, whereas single-agent treatments induce significantly lower levels of DNA damage. Furthermore, we observed the AZD1775/RP6306 combination decreases the expression of the DNA repair protein Rad51 in PalboR cells. To evaluate the clinical potential of dual WEE1 and PKMYT1 inhibition, we generated organoids from metastatic breast cancer lesions of patients who had progressed on CDK4/6 inhibitors for ex vivo studies. Our results demonstrate that dual inhibition of WEE1 and PKMYT1 is more effective than single-agent treatments in eliminating CDK4/6 inhibitor-resistant organoids. Additionally, using patient-derived xenograft mouse models from patients who had progressed on palbociclib and endocrine treatment, we show that the AZD1775/RP6306 combination exhibits superior tumor suppression effects compared to single agents. Overall, this study highlights the potential therapeutic benefits of dual inhibition of WEE1 and PKMYT1 in overcoming CDK4/6 inhibitor resistance in TNBC and ER+ve breast cancer patients. These findings provide a rationale for future clinical trials aimed at exploring the clinical utility of this combination therapy. Citation Format: Mei-Kuang Chen, Grace DeAlessandro, Tuyen Bui, Khandan Keyomarsi. Dual Inhibition of WEE1 and PKMYT1 Synergistically Overcomes CDK4/6 Inhibitor Resistance in Breast Cancer [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 PO2-04-14.

High throughput Targeted Transcriptome Sequencing of Bone Marrow Mononuclear Cells identifies a Rare Leukemia-Specific T Cell Subset.

Abstract Many applications of scRNA-seq are focused on cell type identification, gene regulatory networks, or biomarker discovery. These applications often do not require surveying the whole transcriptome (WT), but rather require the interrogation of specific sets of well-characterized genes. In these cases, sequencing the entire transcriptome may be adding unnecessary costs. To increase throughput and minimize sequencing costs, the development of a targeted gene enrichment method is required. Here, we demonstrate an approach to combine split-pool combinatorial barcoding with enrichment of 1000 genes (Immune1000 panel) representing immune cell markers and pathways. We use this gene panel to characterize 730,000 human bone marrow mononuclear cells (BMMCs) from four leukemia and four healthy donors. We found that enrichment with the Immune1000 Panel enabled the characterization of all major immune cell types at low sequencing depth and allowed for the detection of known leukemia-associated shifts in cell type proportions. Moreover, our targeted sequencing method enabled the detection of a rare (~0.35%), leukemia-specific T cell population that showed highly proliferative and cytotoxic potential. Overall, we demonstrate our modular enrichment strategy preserves biological structure and allows for deep characterization of rare disease-relevant cell types and gene signatures with less sequencing.

Joint single-cell transcriptomic and epigenomic analysis reveals key regulators of CAR T cell stemness and antitumor immunity

Abstract The differentiation of antitumor T cells is tightly controlled by transcriptional and epigenetic pathways. Understanding the programs governing the differentiation of therapeutic T cells such as chimeric antigen receptor (CAR) T cells is crucial for improving their efficacy. Using a single-cell multi-omics approach, we simultaneously profiled chromatin accessibility and transcription within the same single CAR T cell in mice with B-cell leukemia or melanoma. One of the CAR T-cell subsets displayed stem cell-like properties and greater proliferative potential upon tumor challenge. In addition, we uncovered primed chromatin states foreshadowing transcriptional changes and lineage-defining genes heavily regulated at the epigenetic level. By constructing gene regulatory networks in CAR T cells based on the single-cell multi-omics data, we identified hub transcription factors of stem-like CAR T cells including BACH2. By knocking out BACH2 in CAR T cells, we demonstrated an essential role of BACH2 in the development of the stem-like subset and antitumor immunity of CAR T cells. Thus, our study elucidated the transcriptional and epigenetic mechanisms underlying CAR T-cell differentiation and revealed key transcriptional regulators controlling the differentiation and antitumor immunity of CAR T cells.

Functional Phenotype and Ontogeny of Olfr2 Expressing Vascular Macrophages in Atherosclerotic Plaques

Abstract Olfactory receptor 2 (Olfr2) and its human ortholog OR6A2 have emerged as a critical modulator of inflammation, with its presence detected in approximately 30% of vascular macrophages and its depletion shown to reduce atherosclerosis in mice. Single-cell RNA sequencing identified several distinct subsets of macrophages in the atherosclerotic plaque. This study investigates the functional phenotype and ontogeny of Olfr2-expressing vascular macrophages. Olfr2+ macrophage number increased in response to a western diet (WD) in the aorta and they expressed a variety of pro-inflammatory cytokines, including Il1b, and Il6, as well as Ccl5, Cx3cl1, and Ccl22. By extracting differentially expressed genes from bulk RNA-seq data of Olfr2+ vs. Olfr2- vascular macrophages, we defined a signature that mapped with mouse plaque single-cell data of monocytes, aortic intima resident macrophages (MacAir) and Trem2-Gpnmb foamy macrophages. Similar results were found in human plaque, with monocytes and foamy macrophages being significantly enriched. Olfr2 expression also increased in oxLDL-mediated foamy macrophages in vitro. Adoptive transfer experiments confirmed that Olfr2+ macrophages are monocyte-derived, accumulate in the aortic plaque within 72 hours, and display enhanced proliferative potential. Collectively, our findings indicate that Olfr2 expression is boosted by diet in monocytes-derived foamy macrophage subsets that by sensing octanal promote inflammation and atherosclerosis.

MK591 (Quiflapon), a 5-lipoxygenase inhibitor, kills pancreatic cancer cells via downregulation of protein kinase C-epsilon.

Pancreatic tumors and cell lines derived from them exhibit elevated expression of 5-lipoxygenase (5-Lox), whereas non-tumor glands or normal cells do not exhibit this overexpression. Arachidonic acid stimulates pancreatic cancer cell growth via metabolic conversion through the 5-Lox pathway, and inhibition of 5-Lox activity decreases the viability of pancreatic cancer cells. However, the downstream signaling mechanisms through which 5-Lox exerts its effects on the survival of pancreatic cancer cells remain to be elucidated. The effects of 5-Lox inhibition on cell proliferation, apoptosis, and invasive potential were investigated in pancreatic cancer cells. The protein expression was analyzed by Western blot. Apoptosis was analyzed by Annexin-V binding assay and by detecting the degradation of chromatin-DNA to nucleosomal fragments. The protein kinase C-epsilon (PKCε) activity was measured by an immunoprecipitation-kinase assay. The in vivo effects of MK591 were evaluated in pancreatic tumor xenograft model. MK591, a specific inhibitor of 5-Lox activity, killed pancreatic cancer cells via induction of apoptosis, involving externalization of phosphatidylserine, cleavage of PARP (poly-ADP ribose polymerase) and degradation of chromatin DNA to nucleosomes. MK591 effectively blocked in vitro invasion and soft-agar colony formation by pancreatic cancer cells and decreased pancreatic tumor growth in nude mice xenografts. Furthermore, inhibition of 5-Lox downregulated K-Ras and inhibited phosphorylation of c-Raf and ERKs. Interestingly, 5-Lox inhibition induced apoptosis in pancreatic cancer cells without the inhibition of Akt but the protein level of PKCε was dramatically downregulated. Furthermore, inhibition of 5-Lox decreased the phosphorylation of Stat3 at Serine-727. Pre-treatment of pancreatic cancer cells with peptide activators of PKCε prevented apoptosis induced by 5-Lox inhibition, suggesting that the mechanism by which 5-Lox inhibition causes cell death in pancreatic cancer involves downregulation of PKCε. The combination of low doses of MK591 and gemcitabine synergistically reduced the oncogenic phenotype and killed pancreatic cancer cells by inducing apoptosis. These findings indicate that inhibition of 5-Lox interrupts an Akt-independent, PKCε-dependent survival mechanism in pancreatic cancer cells and suggest that metabolism of arachidonic acid through the 5-Lox pathway plays an integral part in the survival of pancreatic cancer cells via signaling through PKCε, an oncogenic, pro-survival serine/threonine kinase.

T-Cell Characteristics Impact Response and Resistance to T-Cell-Redirecting Bispecific Antibodies in Multiple Myeloma.

Bispecific antibodies (BsAb) directed against B-cell maturation antigen (teclistamab) or the orphan G protein-coupled receptor GPRC5D (talquetamab) induce deep and durable responses in heavily pretreated patients with multiple myeloma. However, mechanisms underlying primary and acquired resistance remain poorly understood. The anti-multiple myeloma activity of teclistamab and talquetamab was evaluated in bone marrow (BM) samples from patients with multiple myeloma. T-cell phenotype and function were assessed in BM/peripheral blood samples obtained from patients with multiple myeloma who were treated with these BsAb. In ex vivo killing assays with 41 BM samples from BsAb-naive patients with multiple myeloma, teclistamab- and talquetamab-mediated multiple myeloma lysis was strongly correlated (r = 0.73, P < 0.0001). Both BsAb exhibited poor activity in samples with high regulatory T-cell (Treg) numbers and a low T-cell/multiple myeloma cell ratio. Furthermore, comprehensive phenotyping of BM samples derived from patients treated with teclistamab or talquetamab revealed that high frequencies of PD-1+ CD4+ T cells, CTLA4+ CD4+ T cells, and CD38+ CD4+ T cells were associated with primary resistance. Although this lack of response was linked to a modest increase in the expression of inhibitory receptors, increasing T-cell/multiple myeloma cell ratios by adding extra T cells enhanced sensitivity to BsAb. Further, treatment with BsAb resulted in an increased proportion of T cells expressing exhaustion markers (PD-1, TIGIT, and TIM-3), which was accompanied by reduced T-cell proliferative potential and cytokine secretion, as well as impaired antitumor efficacy in ex vivo experiments. Primary resistance is characterized by a low T-cell/multiple myeloma cell ratio and Treg-driven immunosuppression, whereas reduced T-cell fitness due to continuous BsAb-mediated T-cell activation may contribute to the development of acquired resistance.

Cutibacterium acnes-derived extracellular vesicles promote tumor growth in renal cell carcinoma.

Bacterial flora are present in various parts of the human body, including the intestine, and are thought to be involved in the etiology of various diseases such as multiple sclerosis, intestinal diseases, cancer, and uterine diseases. In recent years, the presence of bacterial 16S rRNA genes has been revealed in blood, which was previously thought to be a sterile environment, and characteristic blood microbiomes have been detected in various diseases. However, the mechanism and the origin of the bacterial information are unknown. In this study, we performed 16S rRNA metagenomic analysis of bacterial DNA in serum extracellular vesicles from five healthy donors and seven patients with renal cell carcinoma and detected Cutibacterium acnes DNA as a characteristic bacterial DNA in the serum extracellular vesicles of patients with renal cell carcinoma. In addition, C. acnes DNA was significantly reduced in postoperative serum extracellular vesicles from patients with renal cell carcinoma compared with that in preoperative serum extracellular vesicles from these patients and was also detected in tumor tissue and extracellular vesicles from tumor tissue-associated microbiota, suggesting an association between C. acnes extracellular vesicles and renal cell carcinoma. C. acnes extracellular vesicles were taken up by renal carcinoma cells to enhance their proliferative potential. C. acnes extracellular vesicles also exhibited tumor-promoting activity in a mouse model of renal cancer allografts with enhanced angiogenesis. These results suggest that extracellular vesicles released by C. acnes localized in renal cell carcinoma tissues act in a tumor-promoting manner.