Enrichment of Melanoma Stem-Like Cells via Sphere Assays

Mukherjee, Nabanita; Lambert, K.; Norris, David A.; Shellman, Yiqun G.

doi:10.1007/978-1-0716-1205-7_14

Cited by 11 publications

(13 citation statements)

References 52 publications

(86 reference statements)

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“…Lentigines of SASH1 S519N (S519N) individuals show an increased population of skin MCs 11 , which could develop via several mechanisms, including increased proliferation or survival of McSCs and/or MCs. Without the availability of stable human McSC cell cultures, we employed a sphere-forming assay with human primary MCs; the sphere assay selects for stem-like characteristics 24 . Compared to the empty vector (EV) control, expressing SASH1 Nonvariant (N.V.) — but not S519N — significantly increased the size and number of formed spheres ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…For sphere forming assays with SASH1 over-expression, 3.75 µg plasmids were introduced into 1.5 × 10 6 HEMn-MP primary MC cells with a Nucleofector 4D system, using the P2 solution and program DS-137 as described 56 . 5 hrs later, cultured cells were detached and either lysed for immunoblot or re-seeded for the sphere-forming 24 or Aldefluor assays 25 . For co-IPs, 2.5 × 10 6 293FT cells were seeded onto 10 cm dishes and grown overnight.…”

Section: Methodsmentioning

confidence: 99%

“…The beads were washed four times, and final lysates were collected with 50 μl elution buffer heated to 70 o C for 15 min. The co-immunoprecipitation lysates were PAGE separated with a 4-12% gel and wet transferred overnight at 30 V. Details on cell culture medium, sphere forming assays, vectors, and antibodies were previously described 24,25 or detailed in the Tables.S1-4.…”

Section: Competing Interestsmentioning

confidence: 99%

“…Three nucleofections were transferred to 6 cm dishes and allowed to recover in medium 254 + HMGS-2 for 5 hours. Cells were detached, and 1 × 10 6 cells were suspended in 25 ml of stem cell medium and seeded in 2 × 10 cm PolyHEMA coated dishes, as in 24 . After 19 hrs in sphere conditions, floating cells were collected and rinsed once with PBS, followed by total RNA extraction with the Qiagen RNeasy Plus Mini kit, including the Qiashredder column.…”

Section: Statistical Analysesmentioning

confidence: 99%

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SASH1 interacts with TNKS2 and promotes human melanocyte stem cell maintenance

Lambert,

Clements,

Mukherjee

et al. 2023

Preprint

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Both aging spots (hyperpigmentation) and hair graying (lack of pigmentation) are associated with aging, two seemingly opposite pigmentation phenotypes. It is not clear how they are mechanistically connected. This study investigated the underlying mechanism in a family with an inherited pigmentation disorder. Clinical examinations identified accelerated hair graying and skin dyspigmentation (intermixed hyper and hypopigmentation) in the family members carrying the SASH1S519Nvariant. Cell assays indicated that SASH1 promoted stem-like characteristics in human melanocytes, and SASH1S519Nwas defective in this function. Multiple assays showed that SASH1 binds to tankyrase 2 (TNKS2), which is required for SASH1’s promotion of stem-like function. Further, the SASH1S519Nvariant is in abona fideTankyrase-binding motif, and SASH1S519Nalters the binding kinetics and affinity. Results here indicate SASH1 as a novel protein regulating the appropriate balance between melanocyte stem cells (McSC) and mature melanocytes (MCs), with S519N variant causing defects. We propose that dysfunction of McSC maintenance connects multiple aging-associated pigmentation phenotypes in the general population.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Competing Interestsmentioning

confidence: 99%

Section: Statistical Analysesmentioning

confidence: 99%

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SASH1 interacts with TNKS2 and promotes human melanocyte stem cell maintenance

Lambert,

Clements,

Mukherjee

et al. 2023

Preprint

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“…The assay was conducted as described previously [ 18 , 49 , 50 , 51 ]. Briefly, cells were seeded at a density of 5k–10k cells/mL with serum-free stem cell media in non-adherent plates.…”

Section: Methodsmentioning

confidence: 99%

A Novel Regimen for Treating Melanoma: MCL1 Inhibitors and Azacitidine

et al. 2021

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Although treatment options for melanoma patients have expanded in recent years with the approval of immunotherapy and targeted therapy, there is still an unmet need for new treatment options for patients that are ineligible for, or resistant to these therapies. BH3 mimetics, drugs that mimic the activity of pro-apoptotic BCL2 family proteins, have recently achieved remarkable success in the clinical setting. The combination of BH3 mimetic ABT-199 (venetoclax) plus azacitidine has shown substantial benefit in treating acute myelogenous leukemia. We evaluated the efficacy of various combinations of BH3 mimetic + azacitidine in fourteen human melanoma cell lines from cutaneous, mucosal, acral and uveal subtypes. Using a combination of cell viability assay, BCL2 family knockdown cell lines, live cell imaging, and sphere formation assay, we found that combining inhibition of MCL1, an anti-apoptotic BCL2 protein, with azacitidine had substantial pro-apoptotic effects in multiple melanoma cell lines. Specifically, this combination reduced cell viability, proliferation, sphere formation, and induced apoptosis. In addition, this combination is highly effective at reducing cell viability in rare mucosal and uveal subtypes. Overall, our data suggest this combination as a promising therapeutic option for some patients with melanoma and should be further explored in clinical trials.

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Learning parameter values of a fractional model of cancer employing boundary densities of tumor cells

Esmaili

2024

Math Methods in App Sciences

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In this paper, a free boundary model of tumor growth with drug application including the heterogeneity or different types of tumor cells (caused by mutations and different values of drug and nutrient concentrations inside the tumor) is studied. Heterogeneity is included in the model by a variable . It is assumed that converting from mutation to mutation happens with probability . A Caputo time fractional‐order hyperbolic equation describes the evolution of tumor cells depending on . It also includes two Caputo time fractional‐order parabolic equations describing the diffusions of nutrients (e.g., oxygen and glucose) and drug concentrations. Instead of integer‐order time derivatives, the fractional ones are considered. In this study, it is aimed to employ the least squares curve fitting method to fit the order of fractional derivatives, coefficient, and rates of the model. For this purpose, using the mathematical model, we have considered the boundary densities of the tumor cells of different types and near‐boundary concentrations of drug and nutrient as the functions of the unknown orders, coefficients and rates (unknown variables). Due to the complexity of the problem, we have obtained the functions numerically. For this, using a change of variable, we have changed the free boundary problem to a problem with fixed domain. Thus, Riemann–Liouville fractional‐order integrals are added to the problem. In the spatial domain, the problem is discretized using the collocation method. In the temporal domain, the fractional derivatives and integrals (with order ) are approximated in mesh points (with step size ) applying a method with error . Then, the unknown variables are obtained by fitting the functions for unknown variables to the data. In order to obtain the variables, a quadratic objective function is considered. The discretized function is substituted into the objective function, then the objective function is minimized using the trust‐region reflective algorithm. Finally, some numerical examples are presented to verify the efficiency of the method. In the examples, we have added noises generated from Gaussian distributions to the data and the effects of noise on the fitted coefficients, and rates are illustrated using some figures and tables. The noisy data are also plotted to have a clear vision of the effects of noises on the data. It is shown that despite the noise, the prediction of the radius of the tumor is acceptable.

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Enrichment of Melanoma Stem-Like Cells via Sphere Assays

Cited by 11 publications

References 52 publications

SASH1 interacts with TNKS2 and promotes human melanocyte stem cell maintenance

SASH1 interacts with TNKS2 and promotes human melanocyte stem cell maintenance

A Novel Regimen for Treating Melanoma: MCL1 Inhibitors and Azacitidine

Learning parameter values of a fractional model of cancer employing boundary densities of tumor cells

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