Single Cell Clones Research Articles

Muscle metabolic stress determines cancer cachexia severity in mice.

Objectives: To determine the metabolic effects of cancer-conditioned media on myotube metabolism and to understand whether the variability of these effects is associated with cancer cachexia progression. Materials and methods: We established single-cell clones from murine Lewis lung carcinoma (LLC) cells and generated conditioned media from each clonal line. Differentiated primary mouse myotubes were incubated with conditioned media derived from each individual clonal cell line. After initial analysis, we selected a specific LLC clonal cell line that failed to induce metabolic stress in myotubes for further investigation in vitro and in vivo. Results: Short-term incubation with conditioned media from 10/34 LLC clonal cells failed to affect oxygen consumption rate (OCR) in myotubes. Incubation with parental LLC-conditioned media decreased protein content and changed the expression of key regulators of muscle function in myotubes, but the incubation of conditioned media from a selected clone that failed to affect OCR in myotubes also did not affect protein content and expression of muscle regulators. Mice injected with parental LLC cells had a significantly reduced body mass and muscle wasting compared to the mice injected with cells derived from this selected LLC clone. Conclusion: Factors secreted by LLC cells induce metabolic stress in primary myotubes and induce cancer cachexia in mice. However, a selected clonal LLC cell line that failed to induce metabolic stress in myotubes also promoted weaker catabolism in mice. These novel findings establish that early disruption of muscle oxidative metabolism is associated with cancer cachexia progression.

CD33 rs12459419 SNP Regulated the Total CD33 Expression Level

Background: Gemutuzumab ozogamicin (GO) is a molecularly targeted drug which combines a humanized anti-CD33 antibody (p67.6) with calicheamicin. GO has differed therapeutic efficacy in cases of acute myeloid leukemia (AML). Single nucleotide polymorphisms (SNPs) in the CD33 gene have been investigated as one of the reasons for this difference. SNPs (CC, CT, and TT types) in rs12459419 have been reported to be associated with clinical outcomes in pediatric AML patients treated with GO-containing chemotherapy. On the other hand, there are reports of poor association in adult AML patients, and the significance of rs12459419 SNPs in remains to be clarified. CD33 is composed of two extracellular domains, IgV and IgC. p67.6, anti-CD33 antibodies, recognize IgV, while HIM3-4 antibodies recognize IgC. rs12459419 SNP is located in the splicing acceptor region of exon 2 of the CD33 gene, when the SNP is T, transcripts that skip exon 2 which encodes the IgV domain, are more likely to occur. The increasing expression of this exon skipping product may be accompanied by a decreasing amount of wild-type CD33 (CD33FL) and an increasing amount of IgV domain-deficient CD33 (CD33ΔE2). GO only recognizes CD33FL, but not CD33ΔE2. We used CRISPR/Cas9 genome editing system to generate cell lines with specifically modified CD33 SNPs to elucidate the expression of CD33FL and CD33ΔE2 by CD33 SNPs and its effect on sensitivity to GO in a uniform genetic background. Methods and Results: First, we analyzed CD33 SNPs in 23 AML patients treated with GO (CC type: 13; CT type; 9: TT type: 1). The outcome of 17 patients, excluding a history of hematopoietic stem cell transplantation before treated with GO, was evaluated. Three-year overall survival rate for the type of CC vs. CT was 70.7% vs. 83.3% (p value = 0.609), and 3-year disease-free survival rate for the type of CC vs. CT was 80.8% vs. 83.3% (p value = 0.895). There was no significant difference in CC and CT. Next, using p67.6 recognizes only CD33FL and HIM3-4 recognizes both CD33FL and CD33ΔE2, we evaluated the expression ratio of CD33FL and CD33ΔE2 in relation with CD33 SNP. We assumed that the expression levels of MFI(median fluorescence intensity) were roughly similar in HIM3-4, but the MFI evaluated by both antibodies tended to be higher in the CT and CC types than in the TT type (MFI for p67.6 of CC, CT and TT was 2675, 1752 and 245, respectively; MFI for HIM3-4 of CC, CT and TT was 699, 629 and 53, respectively). The patient samples differ in many genetic backgrounds besides CD33 SNPs, making accurate evaluation difficult. Genome editing was performed by co-transfection of guide RNAs targeting CD33 SNPs, Cas9 protein and with single-stranded oligonucleotides to the target leukemia cell line. Single cell cloning was performed by limiting dilution to generate single nucleotide altered cells for the CD33 SNP. OCI-AML3 wild-type (TT type) and the obtained CD33 SNP-modified clones (CC type, 4 clones and CT type, 4 clones) were evaluated by flow cytometry with p67.6 and HIM3-4.The results showed that the MFI of both antibodies was higher in the CT and CC types than in the TT, the wild type (MFI for p67.6 of CC, CT and TT was 10760, 13686 and 4801, respectively; MFI for HIM3-4 of CC, CT and TT was 3115, 4067 and 887, respectively). These results showed similar trends to the clinical samples implying that differences in CD33 SNPs affect the amount of total CD33 expression on the cell surface itself. Conclusion: The CD33 rs12459419 SNP regulated the expression level of total CD33 in clinical samples and gene-edited cell lines. There was no obvious difference in the expression levels of total CD33 and CD33FL at the cell surface between the CC and CT types, but it was higher than that of the TT type. The difference in CD33 expression may influence the therapeutic efficacy of GO.

Modeling Flotetuzumab-Associated CRS in AML Using inVitro and inVivo Preclinical Models

Introduction: Flotetuzumab (FLZ) is a bispecific CD123 x CD3 DART® molecule that is being investigated for the treatment of relapsed and refractory acute myeloid leukemia (AML). FLZ induces artificial immunological synapses between CD3 on cytotoxic T-cells and CD123, which is expressed on AML blasts along with off-target hematopoietic and non-hematopoietic tissues. T-cell activation and expansion requires cytokine release from both innate and adaptive immune cells, which when excessive, can result in cytokine release syndrome (CRS), a potentially life-threatening toxicity that manifests with fever, hypotension, organ dysfunction, respiratory failure, and/or coagulopathy. FLZ-mediated anti-leukemia efficacy is often limited by CRS. Therefore, developing pre-clinical models by which to better understand the mechanisms of CRS induction, including collaborating cell types that produce IL-6 and other inflammatory cytokines, is essential to understanding the biology of CRS and developing mechanistically plausible preventative and treatment strategies. MacroGenics has developed a murine genetic knock-in model that expresses a chimeric murine CD3 epsilon (CD3e) chain bearing the human CD3e epitope recognized by FLZ (hCD3e). These mice are immune competent, maintain normal signaling through the T-cell receptor and expand and secrete cytokines upon binding to FLZ and human CD123. To validate this model, we explore whether T-cells harvested from the spleens of immune competent C57Bl/6 hCD3e mice will bind FLZ and kill human CD123-expressing target cells in vitro. Methods: A murine AML cell line (AML1-GFP FLT3-ITD, DNMT3AR878H) was transduced with human CD123 (hCD123) using a lentiviral vector construct and single cell clones expressing high levels of hCD123 were expanded (AMLGFP-hCD123). Spleens from hCD3eHOM C57Bl/6 mice were harvested for isolation of T-lymphocytes via negative immunomagnetic selection on an AutoMACS machine. In vitro killing assays were set up using purified C57Bl/6 hCD3eHOM T-cells and two clones of AMLGFP-hCD123 cells. The effector to target ratio was 1:1, with 2.5x105 cells per well in a 24-well plate. Flow cytometry/FACS was then performed at 24 hours to assess killing of AMLGFP-hCD123 clones 4 and 14 and T-cell subset number and phenotype. Antibodies to the following antigens were used for FACS: mCD4, hCD3 (epitope SP38), mCD25, mCD8, mCD44, mCD62L, hCD123, mCD3, mCD127, mCD69, and mCD45, in addition to endogenous GFP. Results: At 24 hours, target cell killing of clones 4 and 14 was noted (Figure 1). Compared to the controls treated with FLZ or T-cells alone, targets treated with T-cells + FLZ showed a 2.3- and 2.5-fold decrease in the number of AMLGFP-hCD123 targets in clones 4 and 14, respectively, at 24 hours (Figure 1). In terms of T-cell phenotypic changes, the percentage and MFI expression of activation markers, namely CD25 and CD69, increased for CD4+ and CD8+ subsets in the treatment group compared to controls (data not shown). T-cell subsets were characterized as naïve (CD62L+/CD44-), central memory (CD62L+/CD44+), and effector memory (CD62L-/CD44+), as shown in Figure 2. For CD4+ and CD8+ subsets co-cultured with targets, the addition of FLZ induced phenotypic changes away from naïve and towards central and effector memory subsets (Figure 2). In the two control groups, naïve T-cells predominated at the 24-hour time point (Figure 2). Conclusions: The in vitro killing assay shows that T-cells from the spleens of immune competent C57Bl/6 hCD123eHOM mice recognize FLZ and hCD123 on transduced murine leukemia cells. Co-culture with T-cells + target cells + FLZ showed killing at 24 hours compared to controls. T-cell activation and proliferation were noted based on cell surface markers. The shift in phenotype from naïve to central and effector memory subsets suggests T-cell expansion upon leukemia antigen recognition and portends killing of target cells. Pending in vivo studies will evaluate if FLZ can activate endogenous T cells, induce CRS, and kill AMLGFP-hCD123 targets in the huCD3eHOM mice. If successful, we will use the huCD3eHOM model to test the efficacy of various therapies to mitigate CRS and preserve FLZ-mediated T-cell cytotoxicity against AMLGFP-hCD123 targets in huCD3eHOM mice. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Targeting Serine Biosynthesis in Combination with the Modulation of Glutamine Metabolism Enhances Cellular Cytotoxicity in Acute Myeloid Leukemia

Background: Metabolic reprogramming contributes to tumor development and introduces metabolic liabilities that can be exploited to treat cancer. Glutamine (Gln) is the most abundant amino acid intracellularly and in the plasma. Gln depletion has emerged as a therapeutic approach for cancers that have exhibited a dependence on Gln for survival, including acute myeloid leukemia (AML). Crisantaspase, an Erwinia asparaginase, has demonstrated promising anti-AML activity in preclinical/clinical models through interference with plasma Gln supply; however, residual leukemia burden was shown in vivo following Gln depletion by a crisantaspase. Following treatment with asparaginases in vitro, we observed a consistent increase in serine (Ser). Therefore, we hypothesized that in response to Gln depletion, AML cells upregulate de novo Ser biosynthesis as a compensatory pathway, and the inhibition of this pathway will synergize with Gln depletion to increase AML cell death. To test this, we knocked-out phosphoglycerate dehydrogenase (PHGDH), a key enzyme in Ser biosynthesis, in AML cell lines (MOLM-14 and U937) by CRISPR-cas9 and examined the response of the knockout lines to Gln depletion either through Gln withdrawal from the media or crisantaspase treatment. Methods: Single-cell cloning methods were used on bulk populations of PHGDH CRISPR-cas9 knockout cells to characterize positive knockouts and clones were further confirmed by Sangar sequencing. The impact of Gln depletion and PHGDH knockout on AML cell proliferation/viability was assessed using water soluble tetrazolium (WST-1), trypan blue, and measurement of Lactate Dehydrogenase (LDH) release. Immunoblotting was used to examine the expression of the Ser biosynthesis proteins, PHGDH and phosphoserine aminotransferase (PSAT), and glutathione (GSH) levels were measured via a luminescent-based assay. Results: We determined that short-acting crisantaspase (Rylaze) inhibited the proliferation of several AML cell lines with pharmacologically relevant IC50 values ranging from 0.001 to 0.08 µg/mL. Similarly, decreasing the concentration of Gln in the media resulted in a dose-dependent decrease in cell proliferation/growth, confirming AML sensitivity to Gln depletion. Protein expression of PHGDH and PSAT was upregulated following Gln withdrawal from the media (Fig. 1A) as well as following crisantaspase treatment (Fig. 1B). PHGDH knockout AML cells were significantly more sensitive to crisantaspase, yielding IC50 values ranging from 1.34 to 6.59 picogram/mL, ~250-fold lower compared to the parental lines (Fig. 1C). Gln depletion through crisantaspase treatment or withdrawal from the media decreased GSH levels and increased LDH release, both of which were further enhanced by PHGDH knockout. Taken together, these results support dual targeting of Gln and Ser metabolism in AML. Preliminary testing of an NAD-dependent cell permeable PHGDH competitive inhibitor prodrug (Ethyl 2-{4-[(1S)-1-[(4,5-dichloro-1,6-dimethyl-1H-indol-2-yl)formamido]-2-hydroxyethyl]benzenesulfonyl}acetate) demonstrated an inhibition of AML cell proliferation in multiple cell lines, with IC50 concentrations ranging between 1.2 to 2.3 µM. Conclusion: Simultaneous targeting of Ser and Gln metabolism for AML treatment shows promising results in vitro with great potential for translation into clinical practice with clinically available asparaginases, and other glutamine modulators. Ongoing work is focused on the design of a more potent pharmacologic inhibitor of PHGDH to combine with crisantaspase as a novel treatment approach for AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

An unusual presentation of a secondary extramedullary plasmacytoma

Plasma cell dyscrasias are a group of entities characterized by neoplastic proliferation of a single clone of plasma cells, typically producing a monoclonal Immunoglobulin [1].

Establishment and characterization of an immortalized epithelial cell line from human gallbladder.

Although a plethora of studies have employed multiple gallbladder cancer (GBC) cell lines, it is surprisingly noted that there is still lack of a normal gallbladder epithelial cell line as a normal counterpart, thus impeding substantially the progress of mechanistic studies on the transformation of normal epithelial cells to cancer. Here, we created a normal gallbladder epithelial cell line named L-2F7 from human gallbladder tissue. Gallbladder tissues from a diagnosed cholecystitis female patient were collected, and epithelial cells were enriched by magnetic cell sorting. Then, the cells were immortalized by co-introduction of human telomerase reverse transcriptase (hTERT) and Simian virus 40 large T antigen (LT-SV40) via a lentivirus infection system. After clonal selection and isolation, L-2F7 cells were tested for epithelial markers CK7, CK19, CK20, and CD326, genomic feature, cell proliferation, and migration using Western blot, immunofluorescence, whole genome sequencing, karyotyping, and RNA sequencing. L-2F7 cells were also transplanted to Nude (nu/nu) mice to determine tumorigenicity. We successfully identified one single-cell clone named L-2F7 which highly expressed epithelial markers CD326, CK7, CK19, and CK20. This cell line proliferated with a doubling time of 23 h and the epithelial morphology sustained over 30 passages following immortalization. Transient gene transduction of L-2F7 cells led to expression of exogenous GFP and FLAG protein. L-2F7 cells exhibited both distinct non-synonymous mutations from those of gallbladder cancer tissues and differential non-cancerous gene expression patterns similar to normal tissue. Although they displayed unexpected mobility, L-2F7 cells still lacked the ability to develop tumors. We developed a non-cancerous gallbladder epithelial cell line, offering a valuable system for the study of gallbladder cancer and other gallbladder-related disorders.

Efficient and safe single-cell cloning of human pluripotent stem cells using the CEPT cocktail.

Human pluripotent stem cells (hPSCs) are inherently sensitive cells. Single-cell dissociation and the establishment of clonal cell lines have been long-standing challenges. This inefficiency of cell cloning represents a major obstacle for the standardization and streamlining of gene editing in induced pluripotent stem cells for basic and translational research. Here we describe a chemically defined protocol for robust single-cell cloning using microfluidics-based cell sorting in combination with the CEPT small-molecule cocktail. This advanced strategy promotes the viability and cell fitness of self-renewing stem cells. The use of low-pressure microfluidic cell dispensing ensures gentle and rapid dispensing of single cells into 96- and 384-well plates, while the fast-acting CEPT cocktail minimizes cellular stress and maintains cell structure and function immediately after cell dissociation. The protocol also facilitates clone picking and produces genetically stable clonal cell lines from hPSCs in a safe and cost-efficient fashion. Depending on the proliferation rate of the clone derived from a single cell, this protocol can be completed in 7-14 d and requires experience with aseptic cell culture techniques. Altogether, the relative ease, scalability and robustness of this workflow should boost gene editing in hPSCs and leverage a wide range of applications, including cell line development (e.g., reporter and isogenic cell lines), disease modeling and applications in regenerative medicine.

S2556 Not Your Everyday Duodenal Ulcer: Massive GI Bleed Secondary to Extramedullary Plasma Cell Myeloma

Introduction: Plasma cell neoplasms are defined by a monoclonal proliferation of a single clone of immunoglobulin producing plasma cells. Extramedullary plasmacytomas are rare plasma cell dyscrasias that arise outside of the bone marrow, most commonly in the head or neck. Extramedullary multiple myeloma (MM) involves the gastrointestinal (GI) tract in < 5% of cases, and often presents with non-specific symptoms. Here we discuss one such case of uncommon GI involvement of a plasma cell tumor. Case Description/Methods: A 62-year-old male with active lambda light chain MM presented with global fatigue, melena, and hematemesis. Initial vitals included blood pressure 90/62 mmHg and heart rate 95 bpm, with labs revealing normocytic, normochromic anemia with hemoglobin (Hgb) 3.9 g/dL. He was transfused 4 units of packed red blood cells with an appropriate Hgb response to 8.6 g/dL. Immediate esophagogastroduodenoscopy (EGD) found a nonbleeding cratered duodenal ulcer with adherent clot, not amenable to endoscopic intervention (Figure a). He remained hemodynamically stable, and given the lack of continued evidence for GI bleed over the following days, vascular interventional radiology (VIR) embolization was deferred. Prior to discharge, patient was having light brown stools with no active signs of bleeding. He was discharged on pantoprazole, with plan for repeat Hgb within 1 week and repeat EGD in 1 month. Unfortunately, patient was readmitted in cardiac arrest within 24 hours of discharge, with oropharyngeal blood and melena. Resuscitation attempts were unsuccessful. Autopsy determined cause of death to be extramedullary GI disease, with plasma cell infiltration and resultant transmural duodenal ulceration involving the gastroduodenal artery having caused acute hemorrhage (Figureb-c). Discussion: While incredibly rare, due to high mortality and swift progression it is crucial for providers to consider GI extramedullary disease in patients with a history of plasma cell neoplasm who present with upper GI bleed. This holds especially true in cases where the identified lesion is not amenable to endoscopic therapy. Though our patient underwent appropriate resuscitation and evaluation with apparent resolution of symptoms, he died within a day of discharge. Further workup including advanced imaging studies, such as positron emission tomography/computed tomography (PET-CT), or consideration of VIR embolization (deferred here given clinical cessation of bleeding) could be lifesaving.Figure 1.: (a) Ulcer at the duodenal bulb, completely obstructing duodenum. (b) Gastroduodenal ulcer (brackets) with eroded gastroduodenal artery (arrow). (c) H&E, 2X: artery with overlying fibrin cap (1), and surrounding pancreatic parenchyma (2) with plasma cell infiltrate (3).

An integrated microfluidics platform with high-throughput single-cell cloning array and concentration gradient generator for efficient cancer drug effect screening

BackgroundTumor cell heterogeneity mediated drug resistance has been recognized as the stumbling block of cancer treatment. Elucidating the cytotoxicity of anticancer drugs at single-cell level in a high-throughput way is thus of great value for developing precision therapy. However, current techniques suffer from limitations in dynamically characterizing the responses of thousands of single cells or cell clones presented to multiple drug conditions.MethodsWe developed a new microfluidics-based “SMART” platform that is Simple to operate, able to generate a Massive single-cell array and Multiplex drug concentrations, capable of keeping cells Alive, Retainable and Trackable in the microchambers. These features are achieved by integrating a Microfluidic chamber Array (4320 units) and a six-Concentration gradient generator (MAC), which enables highly efficient analysis of leukemia drug effects on single cells and cell clones in a high-throughput way.ResultsA simple procedure produces 6 on-chip drug gradients to treat more than 3000 single cells or single-cell derived clones and thus allows an efficient and precise analysis of cell heterogeneity. The statistic results reveal that Imatinib (Ima) and Resveratrol (Res) combination treatment on single cells or clones is much more efficient than Ima or Res single drug treatment, indicated by the markedly reduced half maximal inhibitory concentration (IC50). Additionally, single-cell derived clones demonstrate a higher IC50 in each drug treatment compared to single cells. Moreover, primary cells isolated from two leukemia patients are also found with apparent heterogeneity upon drug treatment on MAC.ConclusionThis microfluidics-based “SMART” platform allows high-throughput single-cell capture and culture, dynamic drug-gradient treatment and cell response monitoring, which represents a new approach to efficiently investigate anticancer drug effects and should benefit drug discovery for leukemia and other cancers.

Efficacy of pp65-specific TCR-T cell therapy in treating cytomegalovirus infection after hematopoietic stem cell transplantation.

Cytomegalovirus (CMV) infection remains a major cause of mortality after hematopoietic stem cell transplantation (HSCT). Current treatments, including antiviral drugs and adoptive cell therapy with CMV-specific cytotoxic T lymphocytes (CTLs), only show limited benefits in patients. T-cell receptor (TCR)-T cell therapy offers a promising option to treat CMV infections. Here, using tetramer-based screening and single-cell TCR cloning technologies, we identified various CMV antigen-specific TCRs from healthy donors, and generated TCR-T cells targeting multiple pp65 epitopes corresponding to three major HLA-A alleles. The TCR-T cells showed efficient cytotoxicity toward epitope-expressing target cells in vitro. After transfer into immune-deficient mice bearing pp65+ HLA+ tumor cells, TCR-T cells induced dramatic tumor regression and exhibited long-term persistence. In a phase I clinical trial (NCT04153279), CMV TCR-T cells were applied to treat patients with CMV reactivation after HSCT. Except one patient who withdrew at early treatment stage, all other six patients were well-tolerated and achieved complete response (CR), no more than grade 2 cytokine release syndrome (CRS) and other adverse events were observed. CMV TCR-T cells persisted up to 3months. Among them, two patients have survived for more than 1 year. This study demonstrates the great potential in the treatment and prevention of CMV infection following HSCT or other organ transplantation.

POEMS Syndrome- A Review

This review's objective is to provide you with the most recent data about POEMS syndrome. The authors reviewed, compared, and assessed key findings after conducting a literature search of the accessible sources explaining POEMS syndrome. According to the results of this study, POEMS syndrome is related to a class of diseases called monoclonal gammopathies or plasma cell dyscrasias. The unchecked growth of a single clone (monoclonal) of plasma cells causes an abnormal buildup of M-proteins (also known as immunoglobulin) in the blood, which has a detrimental effect on the quality of life for both the patients and their family. To minimize or even eliminate the symptoms, early and appropriate diagnosis and therapy/Treatment are necessary.

High efficiency sorting and outgrowth for single-cell cloning of mammalian cell lines

Single-cell selection and cloning is required for multiple bioprocessing and cell engineering workflows. Dispensing efficiency and outgrowth were optimized for multiple common suspension (CHO ES, Expi293F, and Jurkat) and adherent (MCF-7, A549, CHO-K1, and HEK293) cell lines. Single-cell sorting using a low pressure microfluidic cell sorter, the WOLF Cell Sorter, was compared with limiting dilution at 0.5 cells/well to demonstrate the increased efficiency of using flow cytometry selection of cells. In this work, there was an average single cell deposition on Day 0 of 89.1% across all the cell lines tested compared to 41.2% when using limiting dilution. After growth for 14 days, 66.7% of single-cell clones sorted with the WOLF Cell Sorter survived and only 23.8% when using limiting dilution. Using the WOLF Cell Sorter for cell line development results in higher viable single-cell colonies and the ability to select subpopulations of single-cells using multiple parameters.

Exogenous cholesterol acquisition signaling in LH-responsive MA-10 Leydig cells and in adult mice.

MA-10 cells, established 4 decades ago from a murine Leydig cell tumor, has served as a key model system for studying steroidogenesis. Despite a precipitous loss in their innate ability to respond to luteinizing hormone (LH), the use of a cell-permeable cAMP analog for induction ensured their continued use. In parallel, a paradigm that serum-free conditions are essential for trophic steroidogenic stimulation was rationalized. Through the selection of LH-responsive single-cell MA-10Slip clones, we uncovered that Leydig cells remain responsive in the presence of serum in vitro and that exogenous cholesterol delivery by lipoproteins provided a significantly elevated steroid biosynthetic response (>2-fold). In scrutinizing the underlying regulation, systems biology of the MA-10 cell proteome identified multiple Rho-GTPase signaling pathways as highly enriched. Testing Rho function in steroidogenesis revealed that its modulation can negate the specific elevation in steroid biosynthesis observed in the presence of lipoproteins/serum. This signaling modality primarily linked to the regulation of endocytic traffic is evident only in the presence of exogenous cholesterol. Inhibiting Rho function in vivo also decreased hCG-induced testosterone production in mice. Collectively, our findings dispel a long-held view that the use of serum could confound or interfere with trophic stimulation and underscore the need for exogenous lipoproteins when dissecting physiological signaling and cholesterol trafficking for steroid biosynthesis in vitro. The LH-responsive MA-10Slip clones derived in this study present a reformed platform enabling biomimicry to study the cellular and molecular basis of mammalian steroidogenesis.

PLASMACYTOMA AND MULTIPLE MYELOMA OF THE ORAL CAVITY: A CASE SERIES

Plasma cell neoplasms represent the proliferation of single clone of plasma cells with production of monoclonal immunoglobulins. This study aims to report a case series of plasmacytoma (PMC) and multiple myeloma (MM) of the oral cavity. Data were retrospectively retrieved and clinical, radiographic, microscopic, immunohistochemistry, and follow-up status were collected. Eight cases were included (3 PMC and 5 MM) with a mean age of 53.6 years old (range, 27-78) and a male predilection. Lesions mostly presented as painful swellings in the mandible. Radiographic aspects evidenced multilocular poorly defined lesions and microscopic examinations showed proliferation of plasma cells with abundant eosinophilic cytoplasm. Immunohistochemistry was positive for plasma cell markers and negative for CD20 and CD3, with kappa light chain monoclonality predominance. Overall survival was poor, and 6 patients died, with a mean follow-up time of 5.7 months (range, 1-22). PMC and MM are aggressive neoplasms with a poor prognosis.

Gemcitabine resistance of pancreatic cancer cells is mediated by IGF1R dependent upregulation of CD44 expression and isoform switching

Chemoresistance in pancreatic cancer cells may be caused by the expansion of inherently resistant cancer cells or by the adaptive plasticity of initially sensitive cancer cells. We investigated how CD44 isoforms switching contributed to gemcitabine resistance. Treating CD44 null/low single-cell clones with increasing amounts of gemcitabine caused an increase in expression of CD44 and development of gemcitabine resistant (GR) cells. Drug sensitivity, invasiveness, and EMT process was evaluated by MTT, Matrigel invasion assays, and western blots. Genetic knockdown and pharmacological inhibitors were used to examine the roles of CD44 and IGF1R in mediating gemcitabine resistance. CD44 promoter activity and its interactive EMT-related transcription factors were evaluated by luciferase reporter assay and chromatin immunoprecipitation assay. Kaplan–Meier curve was created by log-rank test to reveal the clinical relevance of CD44 and IGF1R expression in patients. We found silence of CD44 in GR cells partially restored E-cadherin expression, reduced ZEB1 expression, and increased drug sensitivity. The gemcitabine-induced CD44 expressing and isoform switching were associated with an increase in nuclear accumulation of phosphor-cJun, Ets1, and Egr1 and binding of these transcription factors to the CD44 promoter. Gemcitabine treatment induced phosphorylation of IGF1R and increased the expression of phosphor-cJun, Ets1, and Egr1 within 72 h. Stimulation or suppression of IGF1R signaling or its downstream target promoted or blocked CD44 promoter activity. Clinically, patients whose tumors expressed high levels of CD44/IGF1R showed a poor prognosis. This study suggests that IGF1R-dependent CD44 isoform switching confers pancreatic cancer cells to undergo an adaptive change in response to gemcitabine and provides the basis for improved targeted therapy of pancreatic cancer.

Phenotypic heterogeneity driven by plasticity of the intermediate EMT state governs disease progression and metastasis in breast cancer.

The epithelial-to-mesenchymal transition (EMT) is frequently co-opted by cancer cells to enhance migratory and invasive cell traits. It is a key contributor to heterogeneity, chemoresistance, and metastasis in many carcinoma types, where the intermediate EMT state plays a critical tumor-initiating role. We isolate multiple distinct single-cell clones from the SUM149PT human breast cell line spanning the EMT spectrum having diverse migratory, tumor-initiating, and metastatic qualities, including three unique intermediates. Using a multiomics approach, we identify CBFβ as a key regulator of metastatic ability in the intermediate state. To quantify epithelial-mesenchymal heterogeneity within tumors, we develop an advanced multiplexed immunostaining approach using SUM149-derived orthotopic tumors and find that the EMT state and epithelial-mesenchymal heterogeneity are predictive of overall survival in a cohort of stage III breast cancer. Our model reveals previously unidentified insights into the complex EMT spectrum and its regulatory networks, as well as the contributions of epithelial-mesenchymal plasticity (EMP) in tumor heterogeneity in breast cancer.

Isolation of Stably Transfected Melanoma Cell Clones.

Cell populations that have stable changes in their genomic information are widely used by scientists as a research model. They do not require repeated cell transfection as it can lead to a heterogeneous cell population and variable transfection efficiency, affecting reproducibility. Moreover, they are preferable for large-scale analyses. The generation of stable cell clones is useful for a wide range of applications, such as research on gene functions and recombinant protein production. There are a few methods to obtain a homogenous cell population upon initial transient transfection. Here, we describe the isolation of single cell clones with glass cylinders. Although this method has been known for some time, there are a few crucial steps, and neglecting them may lead to failure. We have successfully used this method to obtain clones stably overexpressing a protein of interest (POI) or with knockout of a gene of interest (GOI). We describe preparation steps such as the optimization of selecting drug concentrations, preparation of glass cylinders, and validation of whether the obtained clones have the desired change in the expression of the GOI by PCR, western blot analysis, immunostaining, or gDNA sequencing (depending on the type of derived clones). We also discuss the phenotypic heterogeneity of well-established cell lines as this might be an issue in obtaining stable cell clones.

Notch, RORC and IL-23 signals cooperate to promote multi-lineage human innate lymphoid cell differentiation

Innate lymphoid cells (ILCs) include cytotoxic natural killer cells and distinct groups of cytokine-producing innate helper cells which participate in immune defense and promote tissue homeostasis. Circulating human ILC precursors (ILCP) able to generate all canonical ILC subsets via multi-potent or uni-potent intermediates according to our previous work. Here we show potential cooperative roles for the Notch and IL-23 signaling pathways for human ILC differentiation from blood ILCP using single cell cloning analyses and validate these findings in patient samples with rare genetic deficiencies in IL12RB1 and RORC. Mechanistically, Notch signaling promotes upregulation of the transcription factor RORC, enabling acquisition of Group 1 (IFN-γ) and Group 3 (IL-17A, IL-22) effector functions in multi-potent and uni-potent ILCP. Interfering with RORC or signaling through its target IL-23R compromises ILC3 effector functions but also generally suppresses ILC production from multi-potent ILCP. Our results identify a Notch->RORC- > IL-23R pathway which operates during human ILC differentiation. These observations may help guide protocols to expand functional ILC subsets in vitro with an aim towards novel ILC therapies for human disease.

Diagnosis of a Rare Tumor “Solitary Plasmacytoma of Bone”

Solitary plasmacytoma of bone (SPB, also called osseous plasmacytoma) is a localized tumor in the bone comprised of a single clone of plasma cells in the absence of other features of MM (Multiple Myeloma). Most patients present with skeletal pain or a pathologic fracture of the affected bone. Patients with vertebral involvement may have severe back pain or neurologic compromise (e.g. cord compression). J MEDICINE 2022; 23: 173-175

Editing MC1R in human melanoma cells by CRISPR/Cas9 and functional analysis.

MC1R (melanocortin 1 receptor) encodes the melanocortin-1 receptor, which can activate intracellular cAMP synthesis under the stimulation of the α-melanocyte stimulating hormone (α-MSH) ligand. Increased cAMP then activates the protein kinase A (PKA) pathway, resulting in the up-regulation of the expression of the microphthalmia-associated transcription factor (MITF) which is a critical regulatory factor of melanin synthesis, and tyrosinase (TYR), the rate-limiting enzyme of melanin synthesis tyrosinase (TYR), and ultimately affects production of eumelanin and pheomelanin, and the coat color phenotype of mammalian species. Previous reports have indicated that the mutation A243T in the transmembrane domain 6 (TM6) of MC1R protein might disrupt the function of MC1R, contributing to the red phenotype in Duroc pig. However, functional analysis of the A243T mutation in MC1R has not yet been carried out. In this study, we attempted to used single-stranded oligo-deoxyribonucleotides (ssODN) as donor templates to introduce the c.727G>A (A243T) mutation into MC1R in human melanoma cell line SK-MEL-2 by CRISPR/Cas9 to analyze its effects on MC1R functions. We found the occurrence of ssODN recombination reached to 10%. Unfortunately, Sanger sequencing MC1R in six single-cell clones revealed that none carried the c.727G>A mutation, but all carried undesired mutations surrounding the target site. Cells transfected with CRISPR/Cas9 plasmids and ssODN presented significantly attenuated cAMP activation, and down-regulated MITF and TYR expression, indicating that the editing MC1R could affect the melanin synthesis function in cells. This study provides a basis for further investigation the mechanism of MC1R mutation on animal coat color.