Quantifying rates of cell migration and cell proliferation in co-culture barrier assays reveals how skin and melanoma cells interact during melanoma spreading and invasion

ABSTRACTMalignant spreading involves the migration of cancer cells amongst other native cell types. For example, in vivo melanoma invasion involves individual melanoma cells migrating through native skin, which is composed of several distinct subpopulations of cells. Here, we aim to quantify how interactions between melanoma and fibroblast cells affect the collective spreading of a heterogeneous population of these cells in vitro. We perform a suite of circular barrier assays that includes: (i) monoculture assays with fibroblast cells; (ii) monoculture assays with SK-MEL-28 melanoma cells; and (iii) a series of co-culture assays initiated with three different ratios of SK-MEL-28 melanoma cells and fibroblast cells. Using immunostaining, detailed cell density histograms are constructed to illustrate how the two subpopulations of cells are spatially arranged within the spreading heterogeneous population. Calibrating the solution of a continuum partial differential equation to the experimental results from the monoculture assays allows us to estimate the cell diffusivity and the cell proliferation rate for the melanoma and the fibroblast cells, separately. Using the parameter estimates from the monoculture assays, we then make a prediction of the spatial spreading in the co-culture assays. Results show that the parameter estimates obtained from the monoculture assays lead to a reasonably accurate prediction of the spatial arrangement of the two subpopulations in the co-culture assays. Overall, the spatial pattern of spreading of the melanoma cells and the fibroblast cells is very similar in monoculture and co-culture conditions. Therefore, we find no clear evidence of any interactions other than cell-to-cell contact and crowding effects.

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Tumor quiescence: elevating SOX2 in diverse tumor cell types downregulates a broad spectrum of the cell cycle machinery and inhibits tumor growth

BMC Cancer ◽

10.1186/s12885-020-07370-7 ◽

2020 ◽

Vol 20 (1) ◽

Author(s):

Ethan P. Metz ◽

Erin L. Wuebben ◽

Phillip J. Wilder ◽

Jesse L. Cox ◽

Kaustubh Datta ◽

...

Keyword(s):

Cell Cycle ◽

Cell Proliferation ◽

Tumor Growth ◽

Tumor Cell ◽

Tumor Cells ◽

Molecular Mechanisms ◽

Cell Types ◽

Cell Cycle Machinery

Abstract Background Quiescent tumor cells pose a major clinical challenge due to their ability to resist conventional chemotherapies and to drive tumor recurrence. Understanding the molecular mechanisms that promote quiescence of tumor cells could help identify therapies to eliminate these cells. Significantly, recent studies have determined that the function of SOX2 in cancer cells is highly dose dependent. Specifically, SOX2 levels in tumor cells are optimized to promote tumor growth: knocking down or elevating SOX2 inhibits proliferation. Furthermore, recent studies have shown that quiescent tumor cells express higher levels of SOX2 compared to adjacent proliferating cells. Currently, the mechanisms through which elevated levels of SOX2 restrict tumor cell proliferation have not been characterized. Methods To understand how elevated levels of SOX2 restrict the proliferation of tumor cells, we engineered diverse types of tumor cells for inducible overexpression of SOX2. Using these cells, we examined the effects of elevating SOX2 on their proliferation, both in vitro and in vivo. In addition, we examined how elevating SOX2 influences their expression of cyclins, cyclin-dependent kinases (CDKs), and p27Kip1. Results Elevating SOX2 in diverse tumor cell types led to growth inhibition in vitro. Significantly, elevating SOX2 in vivo in pancreatic ductal adenocarcinoma, medulloblastoma, and prostate cancer cells induced a reversible state of tumor growth arrest. In all three tumor types, elevation of SOX2 in vivo quickly halted tumor growth. Remarkably, tumor growth resumed rapidly when SOX2 returned to endogenous levels. We also determined that elevation of SOX2 in six tumor cell lines decreased the levels of cyclins and CDKs that control each phase of the cell cycle, while upregulating p27Kip1. Conclusions Our findings indicate that elevating SOX2 above endogenous levels in a diverse set of tumor cell types leads to growth inhibition both in vitro and in vivo. Moreover, our findings indicate that SOX2 can function as a master regulator by controlling the expression of a broad spectrum of cell cycle machinery. Importantly, our SOX2-inducible tumor studies provide a novel model system for investigating the molecular mechanisms by which elevated levels of SOX2 restrict cell proliferation and tumor growth.

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Platelets, Tumor Cell Invasiveness, and Metastasis

Blood ◽

10.1182/blood.v122.21.sci-31.sci-31 ◽

2013 ◽

Vol 122 (21) ◽

pp. SCI-31-SCI-31

Author(s):

Richard O. Hynes ◽

Shahinoor Begum ◽

Myriam Labelle

Keyword(s):

Tumor Cell ◽

Tumor Cells ◽

Conflicts Of Interest ◽

Cell Types ◽

Therapeutic Interventions ◽

Migration And Invasion ◽

Cell Contact ◽

Cell Arrest

Abstract Platelets have long been known to promote metastasis, and multiple mechanisms have been proposed to explain this phenomenon, including adhesion, coagulation, and protection against natural killer (NK) cells or turbulence. One mechanism that has been little explored is the possibility that platelets might secrete growth factors or provide other stimuli that could enhance the malignant properties of tumor cells. We have shown that pretreatment of carcinoma cells with platelets induces an EMT-like transformation in their properties in vitro and renders them much more metastatic after introduction into mice. TGF-β, produced by platelets and released on their activation is essential for both the in vitro and the in vivo effects. However, TGF-β alone is insufficient; platelet-tumor cell contact is also required and this contact activates NFkB signaling, which synergizes with the TGF-β signaling. Both signals are required for the enhancement of metastasis. In addition to enhancing migration and invasion in vitro, platelets enhance extravasation in vivo. Earlier work has shown that both P-selectin (expressed on platelets) and L-selectin (expressed on leukocytes) are essential for efficient metastasis, and aggregates of tumor cells, platelets, and leukocytes can be observed at sites of tumor cell arrest and extravasation. It has also been demonstrated by others that leukocytes can enhance extravasation and metastatic seeding. Therefore, we have been interested in the question of the relative roles of platelets and leukocytes in these processes. Which cell types are recruited at the sites of metastatic seeding? Does one cell type depend on another? Which cell types enhance metastasis? What roles do the platelets play in recruiting the other cell types? The involvement of platelets in enhancing metastasis also raises questions about the effects of platelets on circulating tumor cells (CTCs). Could platelets enhance the metastatic capacity of CTCs? Could it be the case that only those CTCs that are associated with platelets and/or leukocytes are functionally involved in seeding metastases? Such aggregates are not scored in most current assays for CTCs and will require new investigative approaches. Platelet participation in metastasis also raises the possibility of therapeutic interventions targeting platelet-specific targets and the paracrine interactions between them and other cells. Disclosures: No relevant conflicts of interest to declare.

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Effects of IL8 and immune cells on the regulation of luteal progesterone secretion

Reproduction ◽

10.1530/rep-13-0602 ◽

2014 ◽

Vol 148 (1) ◽

pp. 21-31 ◽

Cited By ~ 14

Author(s):

Heather Talbott ◽

Abigail Delaney ◽

Pan Zhang ◽

Yangsheng Yu ◽

Robert A Cushman ◽

...

Keyword(s):

Mononuclear Cells ◽

Immune Cell ◽

Neutrophil Migration ◽

Prostaglandin F2α ◽

Cell Types ◽

Luteal Cell ◽

Fibroblast Cells ◽

Progesterone Synthesis

Recent studies have suggested that chemokines may mediate the luteolytic action of prostaglandin F2α (PGF). Our objective was to identify chemokines induced by PGFin vivoand to determine the effects of interleukin 8 (IL8) on specific luteal cell typesin vitro. Mid-cycle cows were injected with saline or PGF, ovaries were removed after 0.5–4 h, and expression of chemokine was analyzed by qPCR.In vitroexpression of IL8 was analyzed after PGF administration and with cell signaling inhibitors to determine the mechanism of PGF-induced chemokine expression. Purified neutrophils were analyzed for migration and activation in response to IL8 and PGF. Purified luteal cell types (steroidogenic, endothelial, and fibroblast cells) were used to identify which cells respond to chemokines. Neutrophils and peripheral blood mononuclear cells (PBMCs) were cocultured with steroidogenic cells to determine their effect on progesterone production.IL8,CXCL2,CCL2, andCCL8transcripts were rapidly increased following PGF treatmentin vivo. The stimulatory action of PGF onIL8mRNA expressionin vitrowas prevented by inhibition of p38 and JNK signaling. IL8, but not PGF, TNF, or TGFB1, stimulated neutrophil migration. IL8 had no apparent action in purified luteal steroidogenic, endothelial, or fibroblast cells, but stimulated ERK phosphorylation in neutrophils. In coculture experiments neither IL8 nor activated neutrophils altered basal or LH-stimulated luteal cell progesterone synthesis. In contrast, activated PBMCs inhibited LH-stimulated progesterone synthesis from cultured luteal cells. These data implicate a complex cascade of events during luteolysis, involving chemokine signaling, neutrophil recruitment, and immune cell action within the corpus luteum.

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Nuclear Localization of BRAFV600E Is Associated with HMOX-1 Upregulation and Aggressive Behavior of Melanoma Cells

Cancers ◽

10.3390/cancers14020311 ◽

2022 ◽

Vol 14 (2) ◽

pp. 311

Author(s):

Mourad Zerfaoui ◽

Eman Toraih ◽

Emmanuelle Ruiz ◽

Youssef Errami ◽

Abdallah S. Attia ◽

...

Keyword(s):

Cell Proliferation ◽

Nuclear Localization ◽

Melanoma Cell ◽

Braf Inhibitor ◽

Melanoma Cells ◽

Heme Oxygenase 1 ◽

Biological Targets ◽

Melanoma Cell Proliferation

Background: Previously, we have demonstrated that nuclear BRAFV600E is associated with melanoma aggressiveness and vemurafenib resistance. However, the underlying mechanisms of how nuclear localization of BRAFV600E promotes cell aggressiveness have not yet been investigated. Despite therapeutic advancements targeting cutaneous melanoma, unknown cellular processes prevent effective treatment for this malignancy, prompting an urgent need to identify new biological targets. This study aims to explore the association of inducible heme oxygenase 1 (HMOX-1) with nuclear BRAFV600E in promoting melanoma aggressiveness. Methods: Proteomics analysis was performed to identify the interacting partner(s) of nuclear BRAFV600E. Immunohistochemistry was applied to evaluate the levels of HMOX-1 and nuclear BRAFV600E expression in melanoma and adjacent healthy tissues. Immunofluorescence assessed the nuclear localization of BRAFV600E in vemurafenib-resistant A375R melanoma cells. Further study of HMOX-1 knockdown or BRAFV600E overexpression in melanoma cells suggested a role for HMOX-1 in the regulation of cell proliferation in vivo and in vitro. Finally, Western blot analysis was performed to confirm the pathway by which HMOX-1 mediates Akt signaling. Results: Proteomics results showed that HMOX-1 protein expression was 10-fold higher in resistant A375R cells compared to parental counterpart cells. In vitro and in vivo results illustrate that nuclear BRAFV600E promotes HMOX-1 overexpression, whereas HMOX-1 reduction represses melanoma cell proliferation and tumor growth. Mechanistic studies revealed that HMOX-1 was associated with nuclear BRAFV600E localization, thus promoting melanoma proliferation via a persistent activation of the AKT pathway. Conclusions: Our results highlight a previously unknown mechanism in which the nuclear BRAFV600E/HMOX-1/AKT axis plays an essential role in melanoma cell proliferation. Targeting HMOX-1 could be a novel method for treating melanoma patients who develop BRAF inhibitor resistance.

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The Immunomodulatory Properties of Amniotic Cells

Cell Transplantation ◽

10.1177/0963689717742819 ◽

2018 ◽

Vol 27 (1) ◽

pp. 31-44 ◽

Cited By ~ 26

Author(s):

Marta Magatti ◽

Elsa Vertua ◽

Anna Cargnoni ◽

Antonietta Silini ◽

Ornella Parolini

Keyword(s):

Cell Proliferation ◽

Cell Types ◽

Culture Conditions ◽

Immunostimulatory Activity ◽

Specific Culture ◽

The Many ◽

Regulatory Functions ◽

Amniotic Cells

Among the many cell types useful in developing therapeutic treatments, human amniotic cells from placenta have been proposed as valid candidates. Both human amniotic epithelial and mesenchymal stromal cells, and the conditioned medium generated from their culture, exert multiple immunosuppressive activities. Indeed, they inhibit T and B cell proliferation, suppress inflammatory properties of monocytes, macrophages, dendritic cells, neutrophils, and natural killer cells, while promoting induction of cells with regulatory functions such as regulatory T cells and anti-inflammatory M2 macrophages. These properties have laid the foundation for their use for the treatment of inflammatory-based diseases, and encouraging results have been obtained in different preclinical disease models where exacerbated inflammation is present. Moreover, an immune-privileged status of amniotic cells has been often highlighted. However, even if long-term engraftment of amniotic cells has been reported into immunocompetent animals, only few cells survive after infusion. Furthermore, amniotic cells have been shown to be able to induce immune responses in vivo and, under specific culture conditions, they can stimulate T cell proliferation in vitro. Although immunosuppressive properties are a widely recognized characteristic of amniotic cells, immunogenic and stimulatory activities appear to be less reported, sporadic events. In order to improve therapeutic outcome, the mechanisms responsible for the suppressive versus stimulatory activity need to be carefully addressed. In this review, both the immunosuppressive and immunostimulatory activity of amniotic cells will be discussed.

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Prevention of spinal neural tube defects in the mouse embryo by growth retardation during neurulation

Development ◽

10.1242/dev.104.2.297 ◽

1988 ◽

Vol 104 (2) ◽

pp. 297-303 ◽

Cited By ~ 1

Author(s):

A.J. Copp ◽

J.A. Crolla ◽

F.A. Brook

Keyword(s):

Cell Proliferation ◽

Neural Tube ◽

Neural Tube Defects ◽

Growth Retardation ◽

Cell Types ◽

Growing Cell ◽

Posterior Neuropore ◽

Curly Tail

Homozygous mutant curly tail mouse embryos developing spinal neural tube defects (NTD) exhibit a cell-type-specific abnormality of cell proliferation that affects the gut endoderm and notochord but not the neuroepithelium. We suggested that spinal NTD in these embryos may result from the imbalance of cell proliferation rates between affected and unaffected cell types. In order to test this hypothesis, curly tail embryos were subjected to influences that retard growth in vivo and in vitro. The expectation was that growth of unaffected rapidly growing cell types would be reduced to a greater extent than affected slowly growing cell types, thus counteracting the genetically determined imbalance of cell proliferation rates and leading to normalization of spinal neurulation. Food deprivation of pregnant females for 48 h prior to the stage of posterior neuropore closure reduced the overall incidence of spinal NTD and almost completely prevented open spina bifida, the most severe form of spinal NTD in curly tail mice. Analysis of embryos earlier in gestation showed that growth retardation acts by reducing the incidence of delayed neuropore closure. Culture of embryos at 40.5 degrees C for 15–23 h from day 10 of gestation, like food deprivation in vivo, also produced growth retardation and led to normalization of posterior neuropore closure. Labelling of embryos in vitro with [3H]thymidine for 1 h at the end of the culture period showed that the labelling index is reduced to a greater extent in the neuroepithelium than in other cell types in growth-retarded embryos compared with controls cultured at 38 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

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Rapid and spontaneous loss of phthiocerol dimycocerosate (PDIM) from Mycobacterium tuberculosis grown in vitro: implications for virulence studies

Microbiology ◽

10.1099/mic.0.029199-0 ◽

2009 ◽

Vol 155 (11) ◽

pp. 3532-3543 ◽

Cited By ~ 88

Author(s):

Pilar Domenech ◽

Michael B. Reed

Keyword(s):

Mycobacterium Tuberculosis ◽

Cell Types ◽

Heterogeneous Population ◽

Short Length ◽

Multiple Mutation ◽

Negative Cell ◽

H37rv Strain ◽

Phthiocerol Dimycocerosate

Isolated in vitro more than half a century ago, the H37Rv strain of Mycobacterium tuberculosis still remains the strain of choice for the majority of laboratories conducting in vivo studies of TB pathogenesis. In this report we reveal that H37Rv is highly prone to losing the ability to synthesize the cell wall lipid phthiocerol dimycocerosate (PDIM) during extended periods of in vitro culture. In addition, H37Rv stocks that have been held in vitro for even a short length of time should be thought of as a heterogeneous population of PDIM-positive and PDIM-negative cell types. We demonstrate that after weekly subculture of PDIM-positive isolates over a period of 20 weeks, the proportion of PDIM-negative cells rises above 30 %. That PDIM biosynthesis is negatively selected in vitro is evident from the broad range of mutation types we observe within cultures originating from a single PDIM-positive parental clone. Moreover, the appearance of these multiple mutation types coupled with an enhanced growth rate of PDIM-negative bacteria ensures that ‘PDIM-less’ clones rapidly dominate in vitro cultures. It has been known for almost a decade that strains of M. tuberculosis that lack PDIM are severely attenuated during in vivo infection. Therefore, the loss of PDIM raises a very serious issue in regard to the interpretation of putative virulence factors where heterogeneous parental cultures are potentially being compared in vivo to recombinant clones isolated within a PDIM-negative background. It is essential that researchers undertaking in vivo virulence studies confirm the presence of PDIM within all recombinant clones and the parental strains they are derived from.

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In vitro Cytotoxicity Studies of Industrially Used Common Nanomaterials on L929 and 3T3 Fibroblast Cells

10.37871/jbres1143 ◽

2020 ◽

Vol 1 (5) ◽

pp. 192-200

Author(s):

Madhulika Srikanth ◽

Waseem S Khan ◽

Ramazan Asmatulu ◽

Heath E Misak ◽

Shang-You Yang ◽

...

Keyword(s):

Mammalian Cells ◽

Cell Types ◽

In Vitro Cytotoxicity ◽

In Vitro Tests ◽

Fibroblast Cells ◽

In Vivo Experiments ◽

Cytotoxicity Studies ◽

Biomedical Electronics

The unique structures and properties of nanomaterials have attracted many engineers and scientists to these resources for different applications, including biomedical, electronics, manufacturing, transportation, energy, and defense. The increasing applications of nanomaterials have also caused some concern among the scientific community about their safety and cytotoxicity. To successfully use nanomaterials in different fields, their interaction with mammalian cells in vitro must be addressed before in vivo experiments can be carried out successfully. In this study, the cytotoxicity values of commonly known nanomaterials, such as 100-ply Carbon Nanotube (CNT) wires, graphene, CNTs, nanoclay, and fullerene, were investigated through in vitro tests on human L929 and mice 3T3 fibroblast cells and compared with each other. The effects of cytotoxicity on both cell types were similar in many ways, but not closely identical due to structural and morphological differences. Compared to mice fibroblast cells, human fibroblast cells have a larger surface area; therefore, the viability values of L929 cells at different dilutions and time durations vary over a larger range. Pristine 100-ply CNT wires were found to be the least cytotoxic, with an average viability of 86.9%, whereas materials with high aspect ratio (e.g., CNTs and graphene) had higher cytotoxicity values due to their potential to pierce through cell membranes.

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Dihydrocapsaicin Inhibits Cell Proliferation and Metastasis in Melanoma via Down-regulating β-Catenin Pathway

Frontiers in Oncology ◽

10.3389/fonc.2021.648052 ◽

2021 ◽

Vol 11 ◽

Author(s):

Shaomin Shi ◽

Chongyang Li ◽

Yanli Zhang ◽

Chaowei Deng ◽

Wei Liu ◽

...

Keyword(s):

Cell Proliferation ◽

Epithelial Mesenchymal Transition ◽

Malignant Tumors ◽

Melanoma Cells ◽

Migration And Invasion ◽

Mice Model ◽

Mesenchymal Transition ◽

Cancer Breast

Dihydrocapsaicin (DHC) is one of the main components of capsaicinoids in Capsicum. It has been reported that DHC exerts anti-cancer effects on diverse malignant tumors, such as colorectal cancer, breast cancer, and glioma. However, studies focused on the effect of DHC upon melanoma have rarely been done. In the present study, melanoma A375 and MV3 cell lines were treated with DHC and the cell proliferation, migration, and invasion were significantly suppressed. Furthermore, DHC effectively inhibited xenograft tumor growth and pulmonary metastasis of melanoma cells in NOD/SCID mice model. It was identified that β-catenin, which plays significant roles in cell proliferation and epithelial-mesenchymal transition, was down-regulated after DHC treatment. In addition, cyclin D1, c-Myc, MMP2, and MMP7, which are critical in diverse cellular process regulation as downstream proteins of β-catenin, were all decreased. Mechanistically, DHC accelerates ubiquitination of β-catenin and up-regulates the beta-transducin repeat containing E3 ubiquitin protein ligase (BTRC) in melanoma cells. The DHC induced suppression of cell proliferation, migration, and invasion were partly rescued by exogenous β-catenin overexpression, both in vitro and in vivo. Taken together, DHC may serve as a candidate natural compound for human melanoma treatment through β-catenin pathway.

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Ultrastructural observations on the in vitro cytoadherence of Plasmodium falciparum infected red blood cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100162132 ◽

1990 ◽

Vol 48 (3) ◽

pp. 914-915

Author(s):

D.J.P. Ferguson ◽

A.R. Berendt ◽

J. Tansey ◽

K. Marsh ◽

C.I. Newbold

Keyword(s):

Plasmodium Falciparum ◽

Red Blood Cells ◽

Blood Cells ◽

Umbilical Vein ◽

Cell Types ◽

Amelanotic Melanoma ◽

Cytoplasmic Process ◽

Umbilical Vein Endothelial Cells

In human malaria, the most serious clinical manifestation is cerebral malaria (CM) due to infection with Plasmodium falciparum. The pathology of CM is thought to relate to the fact that red blood cells containing mature forms of the parasite (PRBC) cytoadhere or sequester to post capillary venules of various tissues including the brain. This in vivo phenomenon has been studied in vitro by examining the cytoadherence of PRBCs to various cell types and purified proteins. To date, three Ijiost receptor molecules have been identified; CD36, ICAM-1 and thrombospondin. The specific changes in the PRBC membrane which mediate cytoadherence are less well understood, but they include the sub-membranous deposition of electron-dense material resulting in surface deformations called knobs. Knobs were thought to be essential for cytoadherence, lput recent work has shown that certain knob-negative (K-) lines can cytoadhere. In the present study, we have used electron microscopy to re-examine the interactions between K+ PRBCs and both C32 amelanotic melanoma cells and human umbilical vein endothelial cells (HUVEC).We confirm previous data demonstrating that C32 cells possess numerous microvilli which adhere to the PRBC, mainly via the knobs (Fig. 1). In contrast, the HUVEC were relatively smooth and the PRBCs appeared partially flattened onto the cell surface (Fig. 2). Furthermore, many of the PRBCs exhibited an invagination of the limiting membrane in the attachment zone, often containing a cytoplasmic process from the endothelial cell (Fig. 2).

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