Cell-free chromatin from dying cancer cells integrate into genomes of bystander healthy cells to induce DNA damage and inflammation

Indraneel Mittra, Urmila Samant, Suvarna Sharma, Gorantla V Raghuram, Tannistha Saha, Pritishkumar Tidke, Namrata Pancholi, Deepika Gupta, Preeti Prasannan, Ashwini Gaikwad, Nilesh Gardi, Rohan Chaubal, Pawan Upadhyay, Kavita Pal, Bhagyeshri Rane, Alfina Shaikh, Sameer Salunkhe, Shilpee Dutt, Pradyumna K Mishra, Naveen K Khare, Naveen K Nair, Amit Dutt, Indraneel Mittra, Urmila Samant, Suvarna Sharma, Gorantla V Raghuram, Tannistha Saha, Pritishkumar Tidke, Namrata Pancholi, Deepika Gupta, Preeti Prasannan, Ashwini Gaikwad, Nilesh Gardi, Rohan Chaubal, Pawan Upadhyay, Kavita Pal, Bhagyeshri Rane, Alfina Shaikh, Sameer Salunkhe, Shilpee Dutt, Pradyumna K Mishra, Naveen K Khare, Naveen K Nair, Amit Dutt

Abstract

Bystander cells of the tumor microenvironment show evidence of DNA damage and inflammation that can lead to their oncogenic transformation. Mediator(s) of cell-cell communication that brings about these pro-oncogenic pathologies has not been identified. We show here that cell-free chromatin (cfCh) released from dying cancer cells are the key mediators that trigger both DNA damage and inflammation in the surrounding healthy cells. When dying human cancer cells were cultured along with NIH3T3 mouse fibroblast cells, numerous cfCh emerged from them and rapidly entered into nuclei of bystander NIH3T3 cells to integrate into their genomes. This led to activation of H2AX and inflammatory cytokines NFκB, IL-6, TNFα and IFNγ. Genomic integration of cfCh triggered global deregulation of transcription and upregulation of pathways related to phagocytosis, DNA damage and inflammation. None of these activities were observed when living cancer cells were co-cultivated with NIH3T3 cells. However, upon intravenous injection into mice, both dead and live cells were found to be active. Living cancer cells are known to undergo extensive cell death when injected intravenously, and we observed that cfCh emerging from both types of cells integrated into genomes of cells of distant organs and induced DNA damage and inflammation. γH2AX and NFκB were frequently co-expressed in the same cells suggesting that DNA damage and inflammation are closely linked pathologies. As concurrent DNA damage and inflammation is a potent stimulus for oncogenic transformation, our results suggest that cfCh from dying cancer cells can transform cells of the microenvironment both locally and in distant organs providing a novel mechanism of tumor invasion and metastasis. The afore-described pro-oncogenic pathologies could be abrogated by concurrent treatment with chromatin neutralizing/degrading agents suggesting therapeutic possibilities.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Cellular uptake and nuclear accumulation of cfCh and their prevention by chromatin neutralizing/degrading agents. (a) Representative laser scanning confocal microscopy images showing nuclear uptake of numerous fluorescent particles by NIH3T3 cells released from dead Jurkat cells pre-labeled with BrdU at 6 h. The uptake of cfCh is prevented by concurrent treatment with CNPs, DNase I and R-Cu. Few nuclear fluorescent signals are seen when live Jurkat cells were used. (b) Quantitative analysis of mean fluorescence intensity (MFI) of images given in a. Fifty nuclei were analyzed for quantifying MFI. Data were analyzed by Student’s t-test. ****P=0.0001. J, Jurkat cells.
Figure 2
Figure 2
Bystander uptake of fluorescently labeled cfCh and activation of DNA damage and inflammation following co-cultivation of NIH3T3 cells with irradiated and un-irradiated GalNAc-T2-GFP HeLa cells. (a) Upper panel, fluorescent microscopy at 36 h showing uptake by bystander NIH3T3 cells of fluorescent cfCh particles released from BrdU pre-labeled dead, but not live, GalNAc-T2-GFP HeLa cells; lower panel, quantitative analysis of mean fluorescence intensity (MFI) of images given in upper panel. (b–e) Upper panels, fluorescent microscopy at 36 h showing activation of H2AX (b), p-ATM (c), NFκB (d) and active Caspase-3 (e) in bystander NIH3T3 cells. Lower panels, quantitative analysis of MFI of images given in upper panel. One thousand nuclei were gated and analyzed for quantifying MFI in each case. *P = 0.05; ****P = 0.0001; NS=Not Significant. Results (mean±S.E.) were analyzed by Student’s t-test. (f) Fluorescent microscopy of NIH3T3 cells co-cultivated with irradiated B16-F10 melanoma cells at 6 h showing BrdU and γ-H2AX co-expressing cells (upper panel); BrdU and IL-6 co-expressing cells (middle panel), and γ-H2AX and IL-6 co-expressing cells (lower panel).
Figure 2
Figure 2
Bystander uptake of fluorescently labeled cfCh and activation of DNA damage and inflammation following co-cultivation of NIH3T3 cells with irradiated and un-irradiated GalNAc-T2-GFP HeLa cells. (a) Upper panel, fluorescent microscopy at 36 h showing uptake by bystander NIH3T3 cells of fluorescent cfCh particles released from BrdU pre-labeled dead, but not live, GalNAc-T2-GFP HeLa cells; lower panel, quantitative analysis of mean fluorescence intensity (MFI) of images given in upper panel. (b–e) Upper panels, fluorescent microscopy at 36 h showing activation of H2AX (b), p-ATM (c), NFκB (d) and active Caspase-3 (e) in bystander NIH3T3 cells. Lower panels, quantitative analysis of MFI of images given in upper panel. One thousand nuclei were gated and analyzed for quantifying MFI in each case. *P = 0.05; ****P = 0.0001; NS=Not Significant. Results (mean±S.E.) were analyzed by Student’s t-test. (f) Fluorescent microscopy of NIH3T3 cells co-cultivated with irradiated B16-F10 melanoma cells at 6 h showing BrdU and γ-H2AX co-expressing cells (upper panel); BrdU and IL-6 co-expressing cells (middle panel), and γ-H2AX and IL-6 co-expressing cells (lower panel).
Figure 3
Figure 3
Detection of fluorescent signals in nuclei of cells of vital organs of mice following i.v. injection of dead and live B16-F10 cells pre-labeled with BrdU and activation of H2AX and NFκB. One hundred thousand BrdU pre-labeled dead and live B16-F10 cells were injected i.v. into mice and animals were killed after 72 h. (a, b) Detection of fluorescent BrdU signals (left hand panels) and activation of H2AX and NFκB (middle panels) in brain, liver and lung following i.v. injection of dead and live B16-F10 cells. It is noteworthy that BrdU signals frequently co-localize with fluorescent signals generated by H2AX and NFκB activation (right hand panels).
Figure 4
Figure 4
Co-localization of γH2AX and NFκB fluorescent signals in vital organs of mice following intravenous injection of dead (upper panels) and live (lower panels) B16-F10 cells. One hundred thousand dead and live B16-F10 cells were injected i.v. into mice and animals were killed after 72 h.
Figure 5
Figure 5
Microarray and pathway analysis of NIH3T3 cells treated with dead Jurkat cells. (a) Heat map of significantly differentially expressed genes in NIH3T3 cells treated with dead Jurkat cells at 0, 2 and 6 h in duplicates. Time points are shown in columns and differentially expressed genes in rows. They were grouped together based on the hierarchical clustering method. Red signifies upregulation status, whereas green signifies downregulation. Black color depicts no change in expression. Scale of heat map shown on the top of the figure. (b–d) Pathway analysis at 6 h showing upregulation of pathways associated with phagocytosis; cell cycle/DNA damage and inflammation, respectively.
Figure 6
Figure 6
Activation of DDR in vitro and in vivo and its inhibition by chromatin neutralizing/degrading agents. (a) Time course of activation of H2AX in NIH3T3 cells following co-cultivation with dead Jurkat cells as detected by indirect immunofluorescence. The experiment was done in duplicate at each time point and 100 cells were analyzed in each case and the average mean fluorescence intensity (MFI) values are depicted in the graph. (b) Prevention of H2AX activation in NIH3T3 cells by CNPs, DNase I and R-Cu following co-cultivation with dead Jurkat cells at 6 h. The experiment was done in duplicate and 500 cells were analyzed in each case for calculating MFI. The histograms depict mean (±S.E.) values in each case. Results were analysed by Student’s t-test. ****P<0.0001. Note that live Jurkat cells do not activate H2AX. (c) Activation of various DDR and DNA repair proteins following co-cultivation of NIH3T3 cells with dead and live Jurkat cells at 6 h as detected by indirect immunofluorescence. The experiments were done in duplicate and 500 cells were analyzed for calculating MFI. The histograms depict mean (±S.E.) values in each case; results were analyzed by Student’s t-test. ****P<0.0001. (d) Activation of H2AX in vital organs and PBMCs following i.v. injection of dead and live B16-F10 cells into mice and their prevention by CNPs, DNase I and R-Cu. One hundred thousand B16-F10 cells were injected i.v. and animals killed after 24 h. The experiments were done in duplicate and 1000 cells were analyzed in each case for calculating MFI. The histograms depict mean (±S.E.) values in each case; results were analyzed by Student’s t-test. *P<0.05, **P<0.01.
Figure 7
Figure 7
Genomic integration of cfCh in vitro and in vivo analyzed by FISH and PCR. (a) Representative FISH images showing presence of abundant human whole-genomic signals in metaphase spreads prepared from NIH3T3 cells co-cultivated with dead Jurkat cells at tenth passage (upper panel). No signals were detected when live Jurkat cells were similarly used. The human whole-genomic probe used did not cross react with mouse DNA. Quantitative analysis of number of human signals per metaphase (lower panel). Thirty metaphases were analyzed in each case. The figure represents mean values (±S.E.). (b) Gel image of PCR-amplified products showing human pan Alu elements in clones E7 and B10. Upper panel, lane 1: 1 kb ladder; lane 2: NIH3T3 cells (negative control); lane 3: human DOK cells (positive control); lane 4 and 5: E7 and B10 clones (respectively); lane 6: no template control. Lower panel, input control PCR, mouse β-ACTIN (104 bp) and human HER2 gene (136 bp) were amplified in mouse NIH3T3 clones and human DOK cells, respectively. (c) Detection of human DNA signals by FISH in vital organs of mice following i.v. injection of dead and live Jurkat cells and their prevention by concurrent treatment with CNPs, DNase I and R-Cu. One hundred thousand Jurkat cells were injected i.v. and animals killed after 7 days. Two animals were used in each case and 500 nuclei were examined for each organ. Percent cells showing human signals were recorded and mean values were compared by Χ2-test. **P<0.01, ****P<0.0001. The human whole-genomic probe used did not cross react with mouse DNA.
Figure 8
Figure 8
Activation of inflammation in vitro and in vivo and its prevention by chromatin neutralizing/degrading agents. (a) Time-course analysis of activation of inflammatory cytokines following co-cultivation of NIH3T3 cells with dead Jurkat cells as detected by indirect immunofluorescence. The experiments were done in duplicates at each time point and the mean values were plotted in the graph. (b) Representative images showing activation of inflammatory cytokines in NIH3T3 cells following co-cultivation with dead and live Jurkat cells with NIH3T3 cells at 6 h and their prevention by CNPs, DNase I and R-Cu. (c) Quantitative analysis of images shown in b. Activation of NFκB (left upper panel), IL-6 (right upper panel), IFNγ (left lower panel) and TNFα (right lower panel). The experiments were done in duplicate and 500 cells were analyzed in each case to determine mean fluorescence intensity (MFI). Mean (±S.E.) values are depicted in the histograms and the results were compared by Student’s t-test. ****P<0.0001. (d–g) Representative images showing activation of inflammatory cytokines at 72 h in vital organs of mice following intravenous injection of dead and live Jurkat cells (1×105) as detected by indirect immunofluorescence. (h) Quantitative analysis of expression of NFκB following intravenous injection of dead and live B16-F10 cells (1×105) and their prevention by CNPs, DNase I and R-Cu. The experiment was done in duplicate and 1000 cells were analyzed in each case to determine MFI. Mean (±S.E.) values are depicted in the histograms and the results were compared by Student’s t-test. **P<0.01, ***P<0.001.
Figure 8
Figure 8
Activation of inflammation in vitro and in vivo and its prevention by chromatin neutralizing/degrading agents. (a) Time-course analysis of activation of inflammatory cytokines following co-cultivation of NIH3T3 cells with dead Jurkat cells as detected by indirect immunofluorescence. The experiments were done in duplicates at each time point and the mean values were plotted in the graph. (b) Representative images showing activation of inflammatory cytokines in NIH3T3 cells following co-cultivation with dead and live Jurkat cells with NIH3T3 cells at 6 h and their prevention by CNPs, DNase I and R-Cu. (c) Quantitative analysis of images shown in b. Activation of NFκB (left upper panel), IL-6 (right upper panel), IFNγ (left lower panel) and TNFα (right lower panel). The experiments were done in duplicate and 500 cells were analyzed in each case to determine mean fluorescence intensity (MFI). Mean (±S.E.) values are depicted in the histograms and the results were compared by Student’s t-test. ****P<0.0001. (d–g) Representative images showing activation of inflammatory cytokines at 72 h in vital organs of mice following intravenous injection of dead and live Jurkat cells (1×105) as detected by indirect immunofluorescence. (h) Quantitative analysis of expression of NFκB following intravenous injection of dead and live B16-F10 cells (1×105) and their prevention by CNPs, DNase I and R-Cu. The experiment was done in duplicate and 1000 cells were analyzed in each case to determine MFI. Mean (±S.E.) values are depicted in the histograms and the results were compared by Student’s t-test. **P<0.01, ***P<0.001.

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Source: PubMed

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