Irradiation-Induced Polyploidy Giant Cancer Cells Mediate Tumor Cell Repopulation via Neosis

2020 ◽  
Author(s):  
Zhengxiang Zhang ◽  
Sijia He ◽  
Feng Xiao ◽  
Jin Cheng ◽  
Yiwei Wang ◽  
...  
Keyword(s):  
Western Blot ◽  
Tumor Cell ◽  
Tumor Cells ◽  
Time Lapse ◽  
Cell Cloning ◽  
X Ray ◽  
Time Lapse Microscopy ◽  
Single Cell Cloning ◽  
Cloning Assay ◽  
Cell Repopulation

Abstract Background Tumor repopulation generally describes the phenomenon that residual tumor cells surviving therapies tenaciously proliferate and reestablish the tumor, presenting an embarrassing plight for cancer treatment. However, the cellular and molecular mechanisms underlying this process remains poorly understood. In this study, we proposed polyploidy giant cancer cells (PGCCs)-mediated and neosis-based tumor repopulation after radiotherapy.Methods The formation of PGCCs after irradiation was examined in vitro and in vivo. The demise of X-ray irradiated cells was detected by flow cytometry, clonogenic cell survival assay and transmission electron microscopy. Western blot was used to test cell proliferation and death related protein expression level of these irradiated cells. Time lapse microscopy was adopted to observe the destiny of PGCCs. The property of these PGCCs was identified by TUNEL assay, Brdu chasing assay, western blot, immunocytochemical and immunofluorescence staining. The relationship of HMGB1 with PGCCs-derived tumor repopulation was conducted via HMGB1 chemical inhibitors. Finally, animal model was used to verify the formation of PGCCs, and the relevance of HMGB1 in this process was investigated by immunohistochemical staining.Results The majority of PGCCs induced by irradiation move towards cell demise, whereas some of them intriguingly possessed proliferative property. Utilizing time-lapse microscopy and single-cell cloning assay, we observed that neosis derived from those PGCCs with proliferative capacity contributed to tumor cell repopulation after irradiation. Using the conditioned media collected from dying tumor cells to perform single-cell cloning assay, we unexpectedly demonstrated that HMGB1 released from dying tumor cells participated the process of neosis-based tumor repopulation. In irradiation treated animal tumor bearing model, the expression level of HMGB1 increased after irradiation compare with non-irradiated group. Moreover, some PGCCs presented high HMGB1 expression. Interestingly, we also observed that the proliferation potential of PGCCs varied. Some PGCCs proliferated at early stage, while some PGCCs proliferated at late stage.Conclusion X-ray irradiation could induce the formation of PGCCs, which could move towards both cell death and survival; irradiation-generating PGCCs mediated tumor cell repopulation after irradiation via neosis; HMGB1 released from dying cells stimulated the process of neosis and participated in tumor repopulation after irradiation.

Temporal Dynamics of Tumor-Microenvironment Interaction and Treatment Responses Revealed through Time-Lapse Compartment-Specific Bioluminescence Imaging: Translational implications

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 276-276
Author(s):  
Eugen Dhimolea ◽  
Emily King ◽  
Efstathios Kastritis ◽  
Douglas W. McMillin ◽  
Jake E. Delmore ◽  
...  
Keyword(s):  
Tumor Cell ◽  
Tumor Cells ◽  
Stromal Cells ◽  
Cell Response ◽  
Temporal Dynamics ◽  
Time Lapse ◽  
Tumor Type ◽  
Time Points ◽  
Cell Responses ◽  
Kinetics Of

Abstract Most conventional methods to sensitively quantify tumor cell proliferation and viability in vitro involve processing of cells in ways that preclude continuation of the respective experiment or prevent the longitudinal collection of data. This common technical feature of conventional assays limits their ability to provide detailed insight into the kinetics of tumor cell responses to treatment(s). Additionally, these limitations hinder the use of these assays to monitor how the kinetics of treatment response can be altered by nonmalignant "accessory" cells of the tumor microenvironment (e.g. bone marrow stromal cells [BMSCs] for hematologic malignancies or bone metastases of solid tumors). To address these obstacles, we modified our previously developed tumor cell compartment-specific bioluminescence imaging (CS-BLI) platform (McMillin et al. Nat Med. 2010), to enable longitudinal assessment of tumor cell response to diverse experimental conditions; we cultured luciferase-expressing tumor cells, with or without stromal cells, in the presence of bioluminescent substrates, using optimized conditions which provide detectable bioluminescent signal even after several days of culture, while having no adverse effect on the viability of tumor or non-malignant cells in this system. This modified approach (time-lapse CSBLI, [TL-CSBLI]) preserved the linear correlation of bioluminescent signal with tumor cell viability. Furthermore, results obtained at the end of the experiment and during interim time-points are consistent with those generated using either non-time-lapse applications of CS-BLI or conventional techniques. We applied TL-CSBLI to delineate, in high-throughput manner, the temporal dynamics of the responses of tumor cells (e.g. multiple myeloma (MM) and other hematologic malignancies) to diverse treatments (e.g. conventional chemotherapeutics, glucocorticoids; proteasome inhibitors (PIs, bortezomib or carfilzomib), and kinase inhibitors). Using the time-lapse capabilities of this assay, we evaluated tumor cell responses in the presence vs. absence of stromal cells. We observed that the kinetics of tumor cell response to diverse therapeutic classes are heterogeneous, even within the same tumor type: for instance, tumor cells with pronounced responses at the end of drug incubation (e.g. 24, 48, 72, hrs after initiation of treatment with PIs, DNA-damaging chemotherapeutics, or dexamethasone respectively), can have different magnitude of responses at intermediate time points. This suggests that TL-CSBLI data can further stratify treatment-responsive tumor cells into those with early vs. late kinetics of response. We also observed that the kinetics of the proliferative / anti-apoptotic effect conferred by stromal cells on tumor cells are highly variable between different cell lines, even within the same tumor type. For instance, the time between initiation of coculture and maximum stimulation of tumor cell viability by stromal cells was variable between cell lines and did not correlate with the magnitude of stimulation by stromal cells. Importantly, TL-CSBLI identified that the response of diverse types of tumor cells to treatments can be delayed in the presence of stromal cells, compared to conventional tumor cell monocultures: this initial delay in treatment response of tumor cells in stromal co-cultures may be observed even in cases where similar cytoreductive responses are eventually observed at later time-points in both the presence and absence of stromal cells. This observation suggests that a more expansive definition of stroma-induced resistance to a given treatment may be warranted, to specifically incorporate the ability of stromal cells to delay the tumor cell response to such treatment. In summary, TL-CSBLI enables detailed characterization of the kinetics of tumor cell responses to diverse experimental conditions. Its use can provide insight into the underappreciated impact that cell-autonomous variations or stroma-induced changes in the kinetics of tumor cell response to a given anti-tumor therapy can have on determining its efficacy. This is particularly consequential for agents (e.g. PIs) which have clinical pharmacokinetic profiles associated with transient peak exposure. Disclosures McMillin: Axios Biosciences: Equity Ownership; DFCI: patent submission on stromal co-culture technologies Patents & Royalties. Negri:DFCI: patent submission on stromal co-culture technologies Patents & Royalties. Mitsiades:Johnson & Johnson: Research Funding; Amgen: Research Funding; Celgene: Consultancy, Honoraria; Millennium Pharmaceuticals: Consultancy, Honoraria; DFCI: patent submission on stromal co-culture technologies Patents & Royalties.

Using a hybrid radioenhancer to realize tumor cell-targeted treatment for osteosarcoma: an in vitro study

2020 ◽  
Vol 27 ◽  
Author(s):  
Fu-I Tung ◽  
Li-Chin Chen ◽  
Yu-Chi Wang ◽  
Ming-Hong Chen ◽  
Pei-Wei Shueng ◽  
...  
Keyword(s):  
Western Blot ◽  
Tumor Cell ◽  
Cell Viability ◽  
Tumor Cells ◽  
Clonogenic Assay ◽  
Normal Cells ◽  
Targeted Radiotherapy ◽  
In Vitro Investigation ◽  
Dose Radiation

: Osteosarcoma is insensitive to radiation. High-dose radiation is often used as a treatment, but causes side effects in patients. Hence, it is important to develop tumor cell-targeted radiotherapy that could improve radiotherapy efficiency on tumor cells and reduce the toxic effect on normal cells during radiation treatment. In this study, we developed an innovative method for treating osteosarcoma by using a novel radiation-enhancer (i.e., carboxymethyl-hexanoyl chitosan-coated selfassembled Au@Fe3O4 nanoparticles; CSAF NPs). CSAF NPs were employed together with 5-aminolevulinic acid (5-ALA) to achieve tumor cell-targeted radiotherapy. In this study, osteosarcoma cells (MG63) and normal cells (MC3T3-E1) were used for an in vitro investigation, in which a reactive oxygen species (ROS) assay, cell viability assay, clonogenic assay, and western blot were used to confirm the treatment efficiency. The ROS assay showed that the combination of CSAF NPs and 5-ALA enhanced radiation-induced ROS production in tumor cells (MG63); however, this was not observed in normal cells (MC3T3-E1). The cell viability ratio of normal cells to tumor cells after treatment with CSAF NPs and 5-ALA reached 2.79. Moreover, the clonogenic assay showed that the radiosensitivity of MG63 cells was increased by the combination use of CSAF NPs and 5-ALA. This was supported by performing a western blot that confirmed expression of cytochrome c (a marker of cell mitochondria damage) and caspase-3 (a marker of cell apoptosis). The results provide an essential basis for developing tumor-cell targeted radiotherapy by means of low-dose radiation.

scholarly journals Bigger, Stronger, Faster: Chimeric Antigen Receptor T Cells Are Olympic Killers

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 814-814 ◽  
Author(s):  
Paul J Neeson ◽  
Alexander James Davenport ◽  
Joseph A Trapani ◽  
Michael Kershaw ◽  
Ryan Cross ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cell ◽  
Tumor Cells ◽  
Chimeric Antigen Receptor ◽  
Antigen Receptor ◽  
Time Lapse ◽  
Immune Synapse ◽  
Car T Cells ◽  
Car T

Abstract Chimeric antigen receptor T cells (CAR T) re-directed to CD19, have induced remarkable responses in clinical trials for patients with B-cell malignancies. Patients have responded to therapy with a CAR T dose which is a fraction of the pre-existing tumor burden. Explanations for this observation include studies which show the proliferative potential of the CAR T cells (Kalos M et al Sci Transl Med 2011), as well as our recent study which showed that individual CAR T cells can serial kill tumor cells (Davenport AJ et al CIR 2015, Figure 6). Using our dual antigen receptor model (OT-I T cell receptor and 2nd generation HER-2 CAR in the same T cell, termed CAR.OTI cells), we also observed a reproducible and significantly shorter time from CAR- vs TCR-mediated activation to detachment from dying tumor cells (Davenport AJ et al CIR 2015, Figure 4D)suggesting that CAR-mediated individual killing events are actually faster. To explore how this may occur, we examined the immune synapse structure at 20 minutes of CAR.OTI CTL co-culture with tumor cells expressing the cognate antigen for either the CAR or TCR. At this timepoint, CAR.OTI CTL, activated via the TCR, formed a conventional bull's eye immune synapse with accumulation of LCK and actin clearance (Figure 1). Surprisingly, CAR.OTI CTL activated via their CAR had an immune synapse with no or diffuse LCK and small actin rings (Figure 1). At the same timepoint, CAR-activated CAR.OTI CTL conjugates with tumor cells were characterized by a microtubule organizing center (MTOC) distant from the immune synapse LCK accumulation. In contrast TCR activated CAR.OTI CTL conjugates consistently had the MTOC proximal to the LCK accumulation (Figure 2A). Despite this, CAR-mediated CAR.OTI CTL killing of tumor targets was inhibited by a protein kinase C zeta inhibitor and is, therefore, MTOC dependent (Figure 2B). The MTOC circumnavigates the activated CTL nucleus and moves to the immune synapse, bringing cytotoxic granules with it. Using time lapse live video (TLLV) microscopy, we compared CAR.OTI CTL cytotoxic granule movement when the CAR.OTI were activated via the TCR vs CAR. We showed that following CAR vs TCR activation, CAR.OTI cytotoxic granules moved with a significantly higher velocity, and had a shorter time lapse to reach the immune synapse following activation (Ca2+ flux), and a significantly shorter time to detachment from the dying tumor cell (Figure 2C). We then re-examined immune synapse formation at an earlier timepoint, to explore whether the data from Figures 1-2 could be explained by a more rapid CTL response following CAR-mediated signaling. In contrast to our observations at 20 minutes, at five minutes we showed CAR-activated CAR.OTI CTL formed conjugates with tumor targets and the immune synapse showed distinct LCK accumulation and actin clearance (Figure 3A). Finally, we explored CAR.OTI CTL signaling and showed that CAR-mediated activation induced a significantly lower number of Ca2+ fluxes, however each Ca2+ flux amplitude was not different (Figure 3B). We also examined changes in proximal (phospho-LCK, pLCK) and distal (phospho-ERK, pERK) signals in CAR- versus TCR- activated CAR.OTI cells, and showed that CAR-mediated activation induced more rapid proximal and distal activation signals (Figure 3C). In conclusion, this study showed that compared to activation by TCR ligation, T cells respond to CAR ligation with faster phospho-protein signaling, Ca2+ flux, formation of an immune synapse and a more rapid movement of the MTOC and delivery of the cytotoxic granules to kill the tumor cells. Furthermore, LFA-1 did not accumulate at the immune synapse following CAR activation, therefore, reduced adhesion may facilitate the observed rapid detachment from the dying tumor cell, and enable the CAR T to rapidly move onto the next tumor target for 'bigger, stronger, faster' killing. Disclosures No relevant conflicts of interest to declare.

Interaction of Human Tumor Cells with Human Platelets and the Coagulation System

1983 ◽  
Vol 50 (03) ◽  
pp. 726-730 ◽  
Author(s):  
Hamid Al-Mondhiry ◽  
Virginia McGarvey ◽  
Kim Leitzel
Keyword(s):  
Tumor Cell ◽  
Tumor Cells ◽  
Cell Lines ◽  
Human Tumor ◽  
Human Platelets ◽  
Factor Vii ◽  
Coagulation System ◽  
Breast Adenocarcinoma ◽  
Activate Factor ◽  
Tumor Cell Lines

SummaryThis paper reports studies on the interaction between human platelets, the plasma coagulation system, and two human tumor cell lines grown in tissue culture: Melanoma and breast adenocarcinoma. The interaction was monitored through the use of 125I- labelled fibrinogen, which measures both thrombin activity generated by cell-plasma interaction and fibrin/fibrinogen binding to platelets and tumor cells. Each tumor cell line activates both the platelets and the coagulation system simultaneously resulting in the generation of thrombin or thrombin-like activity. The melanoma cells activate the coagulation system through “the extrinsic pathway” with a tissue factor-like effect on factor VII, but the breast tumor seems to activate factor X directly. Both tumor cell lines activate platelets to “make available” a platelet- derived procoagulant material necessary for the conversion of prothrombin to thrombin. The tumor-derived procoagulant activity and the platelet aggregating potential of cells do not seem to be inter-related, and they are not specific to malignant cells.

Experience of using time-lapse microscopy in IVF and ICSI programs

2020 ◽  
pp. 47-50
Author(s):  
N. V. Saraeva ◽  
N. V. Spiridonova ◽  
M. T. Tugushev ◽  
O. V. Shurygina ◽  
A. I. Sinitsyna
Keyword(s):  
Pregnancy Rate ◽  
Embryo Transfer ◽  
Selection Procedure ◽  
Time Lapse ◽  
Single Embryo Transfer ◽  
Main Group ◽  
Clinical Pregnancy ◽  
Control Group ◽  
Time Lapse Microscopy

In order to increase the pregnancy rate in the assisted reproductive technology, the selection of one embryo with the highest implantation potential it is very important. Time-lapse microscopy (TLM) is a tool for selecting quality embryos for transfer. This study aimed to assess the benefits of single-embryo transfer of autologous oocytes performed on day 5 of embryo incubation in a TLM-equipped system in IVF and ICSI programs. Single-embryo transfer following incubation in a TLM-equipped incubator was performed in 282 patients, who formed the main group; the control group consisted of 461 patients undergoing single-embryo transfer following a traditional culture and embryo selection procedure. We assessed the quality of transferred embryos, the rates of clinical pregnancy and delivery. The groups did not differ in the ratio of IVF and ICSI cycles, average age, and infertility factor. The proportion of excellent quality embryos for transfer was 77.0% in the main group and 65.1% in the control group (p = 0.001). In the subgroup with receiving eight and less oocytes we noted the tendency of receiving more quality embryos in the main group (р = 0.052). In the subgroup of nine and more oocytes the quality of the transferred embryos did not differ between two groups. The clinical pregnancy rate was 60.2% in the main group and 52.9% in the control group (p = 0.057). The delivery rate was 45.0% in the main group and 39.9% in the control group (p > 0.050).

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