DNA exonuclease Trex1 regulates radiotherapy-induced tumour immunogenicity methods
Aim. Evidence-backed execution summary for DNA exonuclease Trex1 regulates radiotherapy-induced tumour immunogenicity methods from DNA exonuclease Trex1 regulates radiotherapy-induced tumour immunogenicity.
Show snapshot details
On this page
This experiment, in seven questions
Jump straight to the part of the recipe you need. Data and provenance labels stay close to the action they support.
Shopping and prep list
What do I need before I start?
mouse
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Cells and reagents
reagent used in the protocol.
- Use
- BALB/c mouse-derived poorly immunogenic mammary carcinoma 4T1 and TSA cells were obtained from F. Miller and P.L. Lollini, respectively. C57BL/6 mouse-derived poorly immunogenic colorectal carcinoma MCA38 was a gift of Alan Frey. MDA-MB-231 and 4175TR human triple negative breast cancer cells were purchased from A...
Cells and reagents
reagent used in the protocol.
- Use
- Anti-mouse CTLA4 monoclonal antibody (mAb) clone 9H10 (10 mg kg -1; Cat # BE0131), Syrian hamster IgG isotype control (10 mg kg -1; Cat # BE0087), anti-CD8 mAb clone 2.43 (5 mg kg -1; Cat # BE0061) and anti-PD-1 mAb clone RMP1-14 (10 mg kg -1...
In vivo fluorescent imaging
reagent used in the protocol.
- Use
- Mice bearing RFP-tumours were anaesthetized with isoflurane to perform in vivo fluorescent images with IVIS Lumina III in vivo imaging system (Perkin Elmer). The photon radiance on the surface of an animal was expressed as photons per second per centimetre squared per steradian. Images shown are compound pictures ge...
Genome-wide microarray analysis
reagent used in the protocol.
- Use
- TSA tumours growing in WT BALB/c mice were excised 24 h after tumour-directed irradiation with a single dose of 20 Gy or three doses of 8 Gy given in consecutive days. Total RNA was purified with Qiagen RNeasy Mini kit. Quality and quantity of the Total RNA sample was assessed using an Agilent Bioanalyzer with...
Quantification of cytoplasmic dsDNA
reagent used in the protocol.
- Use
- Cytoplasmic extracts from live cells determined by trypan blue exclusion were isolated using NE-PER Nuclear and Cytoplasmic Extraction Reagents (ThermoFisher Scientific, Cat # 78833). dsDNA was quantified in cytoplasmic fraction of 1 × 10 6 live TSA cells 24 h after the last RT exposure using the SpectraMax Qua...
Gene expression analysis
reagent used in the protocol.
- Use
- For analysis of tumour tissue total RNA was extracted after the homogenization of the samples using miRNeasy Mini Kit (QIAGEN, Germany, Cat # 217004) according to the manufacturer's instructions. Extracted RNA was subjected to complementary cDNA synthesis using RT2 preamp cDNA synthesis Kit (Cat # 330401) according...
Gene expression analysis
reagent used in the protocol.
- Use
- For analysis of TSA, MCA38, 4T1, MDA-MB-231, 4175TR cells and their derivatives total RNA was extracted from TRIzol lysates. For experiments testing the induction of Trex1 an initial kinetics analysis showed that Trex1 was upregulated in TSA cells very early following irradiation with 20 Gy reaching a plateau within...
Analysis of IFN-γ production by CD8 + T cells
reagent used in the protocol.
- Use
- Overall, 5 × 10 5 tumour-draining lymph node (TDLN) cells were stimulated ex vivo with 1 µM of the following peptides (GenScript): AH1A5 (SPSYVYHQF), or pMCMV (YPHFMPTNL). AH1A5 is a H2-L d -restricted T cell epitope derived from the gp70 Env product of an endogenous retrovirus, which is an immune-do...
Patient-derived tumour xenograft
Triaged seeds (0.1 × 0.3 × 0.3 cm) from established Patient-derived tumour xenograft (PDTX) from freshly resected primary lung tumour were implanted in a subcutaneous pocket on the flank areas of NOD.Cg-Prkdcscid B2mtm1Unc Il2rgtm1Wjl/SzJ mice (NSG) mice. Tumour growth was evaluated over time by visu...
- Use
- Triaged seeds (0.1 × 0.3 × 0.3 cm) from established Patient-derived tumour xenograft (PDTX) from freshly resected primary lung tumour were implanted in a subcutaneous pocket on the flank areas of NOD.Cg-Prkdcscid B2mtm1Unc Il2rgtm1Wjl/SzJ mice (NSG) mice. Tumour growth was evaluated over time by visu...
In vivo fluorescent imaging
Mice bearing RFP-tumours were anaesthetized with isoflurane to perform in vivo fluorescent images with IVIS Lumina III in vivo imaging system (Perkin Elmer). The photon radiance on the surface of an animal was expressed as photons per second per centimetre squared per steradian. Images shown are compound pictures ge...
- Use
- Mice bearing RFP-tumours were anaesthetized with isoflurane to perform in vivo fluorescent images with IVIS Lumina III in vivo imaging system (Perkin Elmer). The photon radiance on the surface of an animal was expressed as photons per second per centimetre squared per steradian. Images shown are compound pictures ge...
Genome-wide microarray analysis
TSA tumours growing in WT BALB/c mice were excised 24 h after tumour-directed irradiation with a single dose of 20 Gy or three doses of 8 Gy given in consecutive days. Total RNA was purified with Qiagen RNeasy Mini kit. Quality and quantity of the Total RNA sample was assessed using an Agilent Bioanalyzer with...
- Use
- TSA tumours growing in WT BALB/c mice were excised 24 h after tumour-directed irradiation with a single dose of 20 Gy or three doses of 8 Gy given in consecutive days. Total RNA was purified with Qiagen RNeasy Mini kit. Quality and quantity of the Total RNA sample was assessed using an Agilent Bioanalyzer with...
Quantification of cytoplasmic dsDNA
Cytoplasmic extracts from live cells determined by trypan blue exclusion were isolated using NE-PER Nuclear and Cytoplasmic Extraction Reagents (ThermoFisher Scientific, Cat # 78833). dsDNA was quantified in cytoplasmic fraction of 1 × 10 6 live TSA cells 24 h after the last RT exposure using the SpectraMax Qua...
- Use
- Cytoplasmic extracts from live cells determined by trypan blue exclusion were isolated using NE-PER Nuclear and Cytoplasmic Extraction Reagents (ThermoFisher Scientific, Cat # 78833). dsDNA was quantified in cytoplasmic fraction of 1 × 10 6 live TSA cells 24 h after the last RT exposure using the SpectraMax Qua...
Genome-wide microarray analysis
All arrays were processed with Agilent Feature Extraction software and data was analysed with GeneSpring GX software (Agilent Technologies). To compare individual expression values across arrays, raw intensity data was quantile normalized across 18 samples and further normalized to the 0 Gy control for each set. Pro...
- Use
- All arrays were processed with Agilent Feature Extraction software and data was analysed with GeneSpring GX software (Agilent Technologies). To compare individual expression values across arrays, raw intensity data was quantile normalized across 18 samples and further normalized to the 0 Gy control for each set. Pro...
Gene expression analysis
For analysis of TSA, MCA38, 4T1, MDA-MB-231, 4175TR cells and their derivatives total RNA was extracted from TRIzol lysates. For experiments testing the induction of Trex1 an initial kinetics analysis showed that Trex1 was upregulated in TSA cells very early following irradiation with 20 Gy reaching a plateau within...
- Use
- For analysis of TSA, MCA38, 4T1, MDA-MB-231, 4175TR cells and their derivatives total RNA was extracted from TRIzol lysates. For experiments testing the induction of Trex1 an initial kinetics analysis showed that Trex1 was upregulated in TSA cells very early following irradiation with 20 Gy reaching a plateau within...
Flow cytometry analysis
Five days after the last radiation exposure, single cell suspensions obtained from collagenase-digested tumours were stained with the following antibodies purchased from Affymetrix-eBioscience: fixable viability dye efluor 780, CD40-FITC, CD70-APC, CD8α-PE efluor 610, CD45-Alexa fluor 700, CD11c-efluor 45...
- Use
- Five days after the last radiation exposure, single cell suspensions obtained from collagenase-digested tumours were stained with the following antibodies purchased from Affymetrix-eBioscience: fixable viability dye efluor 780, CD40-FITC, CD70-APC, CD8α-PE efluor 610, CD45-Alexa fluor 700, CD11c-efluor 45...
Western blot
Proteins from TSA, 4T1 or MDA-MB-231 parental cells and their derivatives were extracted in RIPA buffer (Sigma, Cat # R0278-50 ml) containing protease inhibitor cocktail (Sigma, Cat # 11836153001). Protein concentrations were determined with BCA method (Thermo Scientific, Cat # 23227). A total of 50 _...
- Use
- Proteins from TSA, 4T1 or MDA-MB-231 parental cells and their derivatives were extracted in RIPA buffer (Sigma, Cat # R0278-50 ml) containing protease inhibitor cocktail (Sigma, Cat # 11836153001). Protein concentrations were determined with BCA method (Thermo Scientific, Cat # 23227). A total of 50 _...
Statistical analysis
Software used for acquisition, scoring, statistics, or reporting.
- Use
- Unless otherwise indicated, data are presented as mean±s.e. of the mean (s.e.m.). For comparisons with only two groups, P values were calculated using unpaired Student's two-tailed t -tests. Survival differences were assessed using log-rank tests. The two-way analysis of variance (ANOVA) test was used for tumou...
Before you run
What should be confirmed before execution?
First confirmation
Equipment is listed but no product mappings are linked.
Confirm before execution
This page is backed by a publishable Replication Data Ledger package with zero critical source-verification issues.
Confirm before execution
Open the source paper before finalizing run-specific details.
Procurement checkpoint
Use source-stated vendors where present. Treat mapped products as sourcing options unless the page marks an exact source match.
Open quote workflowStep-by-step procedure
What do I do, in order?
Radiation dose-dependent IFNβ activation in cancer cells
Having confirmed that 20 Gy and 30 Gy single dose were similarly ineffective, and that abscopal effects with anti-CTLA4 could be observed only after repeated 8 Gy doses, we then studied the differences in tumour response to 20 Gy and 8GyX3. First, gene transcripts induced by radiation in vivo were analysed in tumours shortly after completion of 8GyX3 and 20 Gy, revealing the differential expression of IFN-I stimulated genes (ISGs), upregulated only by 8GyX3 ( ). In vitro irradiation of TSA carcinoma cells in the absence of the tumour stroma showed that the upregulation of ISGs was a cancer cell-intrinsic response, and that only virus infection and 8GyX3 but not 20 Gy could induce the release of IFNβ cytokine by TSA cells ( ). Similar results were obtained with the breast 4T1 and colorectal MCA38 mouse carcinomas, and with the human breast cancer cells MDA-MB-231 ( ). Although the...
Radiation dose dependency of Trex1 induction in cancer cells
To determine if there is a threshold for the induction of Trex1 expression by radiation TSA carcinoma cells were treated with increasing doses of radiation and analysed for cytosolic DNA levels and Trex1 expression. A slight increase in cytosolic dsDNA was measurable in TSA cells treated with 4 Gy and reached a plateau between 8 and 10 Gy radiation ( ). Trex1 levels were significantly increased compared to baseline in cells treated with 12 Gy, and further increased at higher doses, resulting in a significant decrease in cytosolic dsDNA. Similar results were obtained with MCA38 and 4T1 cells, with some differences in the dose threshold at which Trex1 upregulation was sufficient to markedly decrease cytosolic dsDNA, which was 15 Gy in both MCA38 and 4T1 cells ( ). In human breast carcinoma cells, MDA-MB-231 Trex1 expression began to increase only at 15 Gy and reached levels sufficient t...
Radiation dose-dependent IFNβ activation in cancer cells
Induction of IFNAR1 was significantly more robust in TSA cells treated with 8GyX1 and 8GyX3 than 20GyX1 or 30GyX1 radiation ( ). To determine if responsiveness to IFNβ by TSA cells was required for the therapeutic effect of 8GyX3+anti-CTLA4, IFNAR1 expression was abrogated in TSA cells expressing an inducible shRNA targeting Ifnar1 (TSA shIfnar1 ) by feeding mice with doxycycline before tumour irradiation ( ). Regression of the irradiated and abscopal tumours was similar in mice bearing TSA shIfnar1 and control TSA shNS cells at the irradiated site, indicating that responsiveness of cancer cells to IFNβ was not required for 8GyX3+anti-CTLA4-induced tumour inhibition ( ). However, mice with IFNβ-unresponsive TSA cells at the irradiated site mounted weaker tumour-specific CD8 + T cell responses and did not achieve long-term survival due to impaired ability to control the...
Trex1 regulates cytoplasmic DNA levels in irradiated cells
IFNβ production by epithelial cells in the absence of viral infection can be triggered by DNA damage, which leads to accumulation of self-DNA into the cytoplasm. Surprisingly, significantly higher levels of double-stranded DNA (dsDNA) were present in the cytoplasmic fraction of TSA cells treated with one or three 8 Gy radiation doses than with 20 or 30 Gy single doses of radiation ( ). The exonuclease Trex1 plays an essential role in clearance of DNA from the cytoplasm of both haematopoietic and non-haematopoietic cells, and Trex1 gene expression can be upregulated by IFN-stimulatory DNA. To determine if Trex1 could control the abundance of cytoplasmic DNA in TSA cells treated with different radiation doses, its expression was analysed. A significant increase of Trex1 was detected in cells treated with a single dose of 20 and 30 Gy but not 8 Gy, even when given in three consec...
Radiation dose dependency of Trex1 induction in cancer cells
To further confirm the translational relevance of our findings, a patient-derived TP53/KRAS-mutated lung adenocarcinoma xenograft was tested for the ability to upregulate IFN-I pathway and Trex1 gene expression in response to hypofractionated versus high single dose radiation. Similar to the results obtained with the mouse carcinomas, marked upregulation of human Ifnb1 and Mx1 was seen after irradiation with 8 Gy X 1, which was further increased with 8 Gy X 3, while 20 Gy upregulated Trex1 ( ).
Methods
Wild type BALB/c and C57BL/6 mice were purchased from Taconic Animal Laboratory (Germantown, NY, USA). C57BL/6 Ifnar1 -/- mice were purchased from Mutant Mouse Research and Resource Center (MMRRC) at JAX and bred in house. BALB/c Ifnar1 -/- mice were a gift of Dr Joan Durbin, Rutgers, the State University of New Jersey. BALB/c Batf3 -/- mice were purchased from Jackson and bred in house. NOD/SCID/gamma (NOG) female mice (CIEA NOG mouse; NOD.Cg- Prkdc scid Il2rg tm1Sug /JicTac) were purchased from Taconic Animal Laboratory (Germantown, NY, USA). All female and male mice were maintained under pathogen-free conditions in the animal facility at New York University School of medicine and Weill Cornell Medicine and used between 6 and 10 weeks of age. All experiments were approved by the Institutional Animal Care and Use Committee at both institutions.
Cells and reagents
Anti-mouse CTLA4 monoclonal antibody (mAb) clone 9H10 (10 mg kg -1; Cat # BE0131), Syrian hamster IgG isotype control (10 mg kg -1; Cat # BE0087), anti-CD8 mAb clone 2.43 (5 mg kg -1; Cat # BE0061) and anti-PD-1 mAb clone RMP1-14 (10 mg kg -1; Cat # BE0146) were purchased from BioXCell. For experiments with doxycycline, cells were grown in media containing tetracycline-free fetal bovine serum and induced with 4 µg ml -1 of doxycycline 4 days prior to treatment. Recombinant human interleukin-2 (rIL2) was obtained from the Biological Resources Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute.
Tumour challenge and treatment
Perpendicular tumour diameters were measured with a vernier caliper, and tumour volumes were calculated as length × width 2 × 0.52. When applicable, gene knockdown or knock-in expression were induced by adding doxycycline at 100 µg ml -1 (20 mg kg -1 day -1 ) into the mice drinking water at day 8, after tumours establishment. Doxycycline was replenished every 4 days until day 26. On day 12, when primary tumours reached an average size of 60-80 mm 3, animals were randomly assigned to different treatment groups. Radiotherapy (RT) was delivered to the tumour as previously described. Briefly, all mice (including mice receiving sham radiation) were anaesthetized by intra-peritoneal (i.p.) injection of avertin (240 mg kg -1 ) and the primary tumours irradiated with a single fraction of 8 Gy, 20 G...
Measurement outputs
What raw and processed outputs should exist?
Having confirmed that 20 Gy and 30 Gy single dose were similarly ineffective, and that abscopal effects with anti-CTLA4 could be observed only after repeated 8 Gy doses, we then...
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
To determine if there is a threshold for the induction of Trex1 expression by radiation TSA carcinoma cells were treated with increasing doses of radiation and analysed for cyto...
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
Induction of IFNAR1 was significantly more robust in TSA cells treated with 8GyX1 and 8GyX3 than 20GyX1 or 30GyX1 radiation ( ). To determine if responsiveness to IFNβ by T...
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
IFNβ production by epithelial cells in the absence of viral infection can be triggered by DNA damage, which leads to accumulation of self-DNA into the cytoplasm. Surprisin...
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
Analysis plan
How should the outputs become interpretable results?
Acquisition
Capture matched images from the relevant tissue region using the same acquisition settings across samples.
inferred from protocolPreprocessing / cleaning
Having confirmed that 20 Gy and 30 Gy single dose were similarly ineffective, and that abscopal effects with anti-CTLA4 could be observed only after repeated 8 Gy doses, we then studied the differences in tumour response to 20 Gy and 8GyX3.
from paperScoring or quantification
Quantify the primary readouts for this experiment: Having confirmed that 20 Gy and 30 Gy single dose were similarly ineffective, and that abscopal effects with anti-CTLA4 could be observed only after repeated 8 Gy doses, we then...; To determine if there is a threshold for the induction of Trex1 expression by radiation TSA carcinoma cells were treated with increasing doses of radiation and analysed for cyto...; Induction of IFNAR1 was significantly more robust in TSA cells treated with 8GyX1 and 8GyX3 than 20GyX1 or 30GyX1 radiation ( ). To determine if responsiveness to IFNβ by T...; IFNβ production by epithelial cells in the absence of viral infection can be triggered by DNA damage, which leads to accumulation of self-DNA into the cytoplasm. Surprisin....
from paperNormalization
Normalize expression or signal values against the stated control or loading reference before comparing groups.
inferred from protocolStatistical comparison
Having confirmed that 20 Gy and 30 Gy single dose were similarly ineffective, and that abscopal effects with anti-CTLA4 could be observed only after repeated 8 Gy doses, we then...; To determine if there is a threshold for the induction of Trex1 expression by radiation TSA carcinoma cells were treated with increasing doses of radiation and analysed for cyto...; IFNβ production by epithelial cells in the absence of viral infection can be triggered by DNA damage, which leads to accumulation of self-DNA into the cytoplasm. Surprisin...; On the basis of past experience and assuming a normal distribution and a coefficient of variation of approximately 25% for tumour growth rate, we determined that at least seven...
from paperReporting output
Report representative outputs alongside summary comparisons for Having confirmed that 20 Gy and 30 Gy single dose were similarly ineffective, and that abscopal effects with anti-CTLA4 could be observed only after repeated 8 Gy doses, we then..., To determine if there is a threshold for the induction of Trex1 expression by radiation TSA carcinoma cells were treated with increasing doses of radiation and analysed for cyto..., Induction of IFNAR1 was significantly more robust in TSA cells treated with 8GyX1 and 8GyX3 than 20GyX1 or 30GyX1 radiation ( ). To determine if responsiveness to IFNβ by T..., IFNβ production by epithelial cells in the absence of viral infection can be triggered by DNA damage, which leads to accumulation of self-DNA into the cytoplasm. Surprisin....
inferred from protocolStructured statistical methods
Having confirmed that 20 Gy and 30 Gy single dose were similarly ineffective, and that abscopal effects with anti-CTLA4 could be observed only after repeated 8 Gy doses, we then...; To determine if there is a threshold for the induction of Trex1 expression by radiation TSA carcinoma cells were treated with increasing doses of radiation and analysed for cyto...; IFNβ production by epithelial cells in the absence of viral infection can be triggered by DNA damage, which leads to accumulation of self-DNA into the cytoplasm. Surprisin...; On the basis of past experience and assuming a normal distribution and a coefficient of variation of approximately 25% for tumour growth rate, we determined that at least seven...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (8)
Having confirmed that 20 Gy and 30 Gy single dose were similarly ineffective, and that abscopal effects with anti-CTLA4 could be observed only after repeated 8 Gy doses, we then studied the differences in tumour response to 20 Gy and 8GyX3. First, gene transcripts induced by radiation in vivo were analysed in tumours shortly after completion of 8GyX3 and 20 Gy, revealing the differential expression of IFN-I stimulated genes (ISGs), upregulated only by 8GyX3 ( ). In vitro irradiation of TSA carcinoma cells in the absence of the tumour stroma showed that the upregulation of ISGs was a cancer cell-intrinsic response, and that only virus infection and 8GyX3 but not 20 Gy could induce the release of IFNβ cytokine by TSA cells ( ). Similar results were obtained with the breast 4T1 and colorectal MCA38 mouse carcinomas, and with the human breast cancer cells MDA-MB-231 ( ). Although the latter did not show significant increase of Ifnb1 gene expression, increased secretion of IFNβ was observed in response to 8GyX3. Analysis of tumour-infiltrating DCs (TIDCs) revealed a marked increase in CD8α + TIDCs within TSA tumours after 8GyX3 but not 20 Gy radiation, consistent with...
To determine if there is a threshold for the induction of Trex1 expression by radiation TSA carcinoma cells were treated with increasing doses of radiation and analysed for cytosolic DNA levels and Trex1 expression. A slight increase in cytosolic dsDNA was measurable in TSA cells treated with 4 Gy and reached a plateau between 8 and 10 Gy radiation ( ). Trex1 levels were significantly increased compared to baseline in cells treated with 12 Gy, and further increased at higher doses, resulting in a significant decrease in cytosolic dsDNA. Similar results were obtained with MCA38 and 4T1 cells, with some differences in the dose threshold at which Trex1 upregulation was sufficient to markedly decrease cytosolic dsDNA, which was 15 Gy in both MCA38 and 4T1 cells ( ). In human breast carcinoma cells, MDA-MB-231 Trex1 expression began to increase only at 15 Gy and reached levels sufficient to markedly decrease cytosolic dsDNA at 18 Gy ( ), while in human breast carcinoma cells 4175TR a slight increase in Trex1 expression was already detectable at 10 Gy and reached levels sufficient to markedly decrease cytosolic dsDNA at 12 Gy ( ). Thus, in all cancer cells tested high radiation doses...
Induction of IFNAR1 was significantly more robust in TSA cells treated with 8GyX1 and 8GyX3 than 20GyX1 or 30GyX1 radiation ( ). To determine if responsiveness to IFNβ by TSA cells was required for the therapeutic effect of 8GyX3+anti-CTLA4, IFNAR1 expression was abrogated in TSA cells expressing an inducible shRNA targeting Ifnar1 (TSA shIfnar1 ) by feeding mice with doxycycline before tumour irradiation ( ). Regression of the irradiated and abscopal tumours was similar in mice bearing TSA shIfnar1 and control TSA shNS cells at the irradiated site, indicating that responsiveness of cancer cells to IFNβ was not required for 8GyX3+anti-CTLA4-induced tumour inhibition ( ). However, mice with IFNβ-unresponsive TSA cells at the irradiated site mounted weaker tumour-specific CD8 + T cell responses and did not achieve long-term survival due to impaired ability to control the abscopal tumour, which either did not regress completely or recurred, while 43% of the mice with IFNAR1 + TSA cells at the irradiated site remained tumour-free for over 100 days ( ). Thus, IFNβ responsiveness is required in both, the tumour and the host, for optimal induction of anti-tumour imm...
IFNβ production by epithelial cells in the absence of viral infection can be triggered by DNA damage, which leads to accumulation of self-DNA into the cytoplasm. Surprisingly, significantly higher levels of double-stranded DNA (dsDNA) were present in the cytoplasmic fraction of TSA cells treated with one or three 8 Gy radiation doses than with 20 or 30 Gy single doses of radiation ( ). The exonuclease Trex1 plays an essential role in clearance of DNA from the cytoplasm of both haematopoietic and non-haematopoietic cells, and Trex1 gene expression can be upregulated by IFN-stimulatory DNA. To determine if Trex1 could control the abundance of cytoplasmic DNA in TSA cells treated with different radiation doses, its expression was analysed. A significant increase of Trex1 was detected in cells treated with a single dose of 20 and 30 Gy but not 8 Gy, even when given in three consecutive fractions, for a total dose of 24 Gy over 3 days ( ). In contrast, release of IFNβ and expression of ISGs Mx1, Ifnar1 and Cxcl10 were markedly increased only by the 8GyX3 regimen of radiation ( and ). Interestingly, while a single dose of 8 Gy induced expression of IFNAR1 and modest leve...
To further confirm the translational relevance of our findings, a patient-derived TP53/KRAS-mutated lung adenocarcinoma xenograft was tested for the ability to upregulate IFN-I pathway and Trex1 gene expression in response to hypofractionated versus high single dose radiation. Similar to the results obtained with the mouse carcinomas, marked upregulation of human Ifnb1 and Mx1 was seen after irradiation with 8 Gy X 1, which was further increased with 8 Gy X 3, while 20 Gy upregulated Trex1 ( ).
Wild type BALB/c and C57BL/6 mice were purchased from Taconic Animal Laboratory (Germantown, NY, USA). C57BL/6 Ifnar1 -/- mice were purchased from Mutant Mouse Research and Resource Center (MMRRC) at JAX and bred in house. BALB/c Ifnar1 -/- mice were a gift of Dr Joan Durbin, Rutgers, the State University of New Jersey. BALB/c Batf3 -/- mice were purchased from Jackson and bred in house. NOD/SCID/gamma (NOG) female mice (CIEA NOG mouse; NOD.Cg- Prkdc scid Il2rg tm1Sug /JicTac) were purchased from Taconic Animal Laboratory (Germantown, NY, USA). All female and male mice were maintained under pathogen-free conditions in the animal facility at New York University School of medicine and Weill Cornell Medicine and used between 6 and 10 weeks of age. All experiments were approved by the Institutional Animal Care and Use Committee at both institutions.
Anti-mouse CTLA4 monoclonal antibody (mAb) clone 9H10 (10 mg kg -1; Cat # BE0131), Syrian hamster IgG isotype control (10 mg kg -1; Cat # BE0087), anti-CD8 mAb clone 2.43 (5 mg kg -1; Cat # BE0061) and anti-PD-1 mAb clone RMP1-14 (10 mg kg -1; Cat # BE0146) were purchased from BioXCell. For experiments with doxycycline, cells were grown in media containing tetracycline-free fetal bovine serum and induced with 4 µg ml -1 of doxycycline 4 days prior to treatment. Recombinant human interleukin-2 (rIL2) was obtained from the Biological Resources Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute.
Perpendicular tumour diameters were measured with a vernier caliper, and tumour volumes were calculated as length × width 2 × 0.52. When applicable, gene knockdown or knock-in expression were induced by adding doxycycline at 100 µg ml -1 (20 mg kg -1 day -1 ) into the mice drinking water at day 8, after tumours establishment. Doxycycline was replenished every 4 days until day 26. On day 12, when primary tumours reached an average size of 60-80 mm 3, animals were randomly assigned to different treatment groups. Radiotherapy (RT) was delivered to the tumour as previously described. Briefly, all mice (including mice receiving sham radiation) were anaesthetized by intra-peritoneal (i.p.) injection of avertin (240 mg kg -1 ) and the primary tumours irradiated with a single fraction of 8 Gy, 20 Gy or 30 Gy on day 12 or with 3 fractions of 8 Gy on day 12, 13 and 14 using the Small Animal Radiation Research Platform (SARRP Xstrahl, Surrey, UK). Anti-mouse CTLA4 mAb or its isotype control mAbs were administered i.p. (200 µg per mouse) on days 14, 17 and 20. Anti-mouse PD-1 mAb was g...
Machine-readable layer
[
{
"@context": "https://schema.org",
"@type": "HowTo",
"name": "DNA exonuclease Trex1 regulates radiotherapy-induced tumour immunogenicity methods",
"description": "Evidence-backed execution summary for DNA exonuclease Trex1 regulates radiotherapy-induced tumour immunogenicity methods from DNA exonuclease Trex1 regulates radiotherapy-induced tumour immunogenicity.",
"totalTime": "PT77280M",
"step": [
{
"@type": "HowToStep",
"position": 1,
"name": "Radiation dose-dependent IFNβ activation in cancer cells",
"text": "Having confirmed that 20 Gy and 30 Gy single dose were similarly ineffective, and that abscopal effects with anti-CTLA4 could be observed only after repeated 8 Gy doses, we then studied the differences in tumour response to 20 Gy and 8GyX3. First, gene transcripts induced by radiation in vivo were analysed in tumours shortly after completion of 8GyX3 and 20 Gy, revealing the differential expression of IFN-I stimulated genes (ISGs), upregulated only by 8GyX3 ( ). In vitro irradiation of TSA carcinoma cells in the absence of the tumour stroma showed that the upregulation of ISGs was a cancer cell-intrinsic response, and that only virus infection and 8GyX3 but not 20 Gy could induce the release of IFNβ cytokine by TSA cells ( ). Similar results were obtained with the breast 4T1 and colorectal MCA38 mouse carcinomas, and with the human breast cancer cells MDA-MB-231 ( ). Although the..."
},
{
"@type": "HowToStep",
"position": 2,
"name": "Radiation dose dependency of Trex1 induction in cancer cells",
"text": "To determine if there is a threshold for the induction of Trex1 expression by radiation TSA carcinoma cells were treated with increasing doses of radiation and analysed for cytosolic DNA levels and Trex1 expression. A slight increase in cytosolic dsDNA was measurable in TSA cells treated with 4 Gy and reached a plateau between 8 and 10 Gy radiation ( ). Trex1 levels were significantly increased compared to baseline in cells treated with 12 Gy, and further increased at higher doses, resulting in a significant decrease in cytosolic dsDNA. Similar results were obtained with MCA38 and 4T1 cells, with some differences in the dose threshold at which Trex1 upregulation was sufficient to markedly decrease cytosolic dsDNA, which was 15 Gy in both MCA38 and 4T1 cells ( ). In human breast carcinoma cells, MDA-MB-231 Trex1 expression began to increase only at 15 Gy and reached levels sufficient t..."
},
{
"@type": "HowToStep",
"position": 3,
"name": "Radiation dose-dependent IFNβ activation in cancer cells",
"text": "Induction of IFNAR1 was significantly more robust in TSA cells treated with 8GyX1 and 8GyX3 than 20GyX1 or 30GyX1 radiation ( ). To determine if responsiveness to IFNβ by TSA cells was required for the therapeutic effect of 8GyX3+anti-CTLA4, IFNAR1 expression was abrogated in TSA cells expressing an inducible shRNA targeting Ifnar1 (TSA shIfnar1 ) by feeding mice with doxycycline before tumour irradiation ( ). Regression of the irradiated and abscopal tumours was similar in mice bearing TSA shIfnar1 and control TSA shNS cells at the irradiated site, indicating that responsiveness of cancer cells to IFNβ was not required for 8GyX3+anti-CTLA4-induced tumour inhibition ( ). However, mice with IFNβ-unresponsive TSA cells at the irradiated site mounted weaker tumour-specific CD8 + T cell responses and did not achieve long-term survival due to impaired ability to control the..."
},
{
"@type": "HowToStep",
"position": 4,
"name": "Trex1 regulates cytoplasmic DNA levels in irradiated cells",
"text": "IFNβ production by epithelial cells in the absence of viral infection can be triggered by DNA damage, which leads to accumulation of self-DNA into the cytoplasm. Surprisingly, significantly higher levels of double-stranded DNA (dsDNA) were present in the cytoplasmic fraction of TSA cells treated with one or three 8 Gy radiation doses than with 20 or 30 Gy single doses of radiation ( ). The exonuclease Trex1 plays an essential role in clearance of DNA from the cytoplasm of both haematopoietic and non-haematopoietic cells, and Trex1 gene expression can be upregulated by IFN-stimulatory DNA. To determine if Trex1 could control the abundance of cytoplasmic DNA in TSA cells treated with different radiation doses, its expression was analysed. A significant increase of Trex1 was detected in cells treated with a single dose of 20 and 30 Gy but not 8 Gy, even when given in three consec..."
},
{
"@type": "HowToStep",
"position": 5,
"name": "Radiation dose dependency of Trex1 induction in cancer cells",
"text": "To further confirm the translational relevance of our findings, a patient-derived TP53/KRAS-mutated lung adenocarcinoma xenograft was tested for the ability to upregulate IFN-I pathway and Trex1 gene expression in response to hypofractionated versus high single dose radiation. Similar to the results obtained with the mouse carcinomas, marked upregulation of human Ifnb1 and Mx1 was seen after irradiation with 8 Gy X 1, which was further increased with 8 Gy X 3, while 20 Gy upregulated Trex1 ( )."
},
{
"@type": "HowToStep",
"position": 6,
"name": "Methods",
"text": "Wild type BALB/c and C57BL/6 mice were purchased from Taconic Animal Laboratory (Germantown, NY, USA). C57BL/6 Ifnar1 -/- mice were purchased from Mutant Mouse Research and Resource Center (MMRRC) at JAX and bred in house. BALB/c Ifnar1 -/- mice were a gift of Dr Joan Durbin, Rutgers, the State University of New Jersey. BALB/c Batf3 -/- mice were purchased from Jackson and bred in house. NOD/SCID/gamma (NOG) female mice (CIEA NOG mouse; NOD.Cg- Prkdc scid Il2rg tm1Sug /JicTac) were purchased from Taconic Animal Laboratory (Germantown, NY, USA). All female and male mice were maintained under pathogen-free conditions in the animal facility at New York University School of medicine and Weill Cornell Medicine and used between 6 and 10 weeks of age. All experiments were approved by the Institutional Animal Care and Use Committee at both institutions."
},
{
"@type": "HowToStep",
"position": 7,
"name": "Cells and reagents",
"text": "Anti-mouse CTLA4 monoclonal antibody (mAb) clone 9H10 (10 mg kg -1; Cat # BE0131), Syrian hamster IgG isotype control (10 mg kg -1; Cat # BE0087), anti-CD8 mAb clone 2.43 (5 mg kg -1; Cat # BE0061) and anti-PD-1 mAb clone RMP1-14 (10 mg kg -1; Cat # BE0146) were purchased from BioXCell. For experiments with doxycycline, cells were grown in media containing tetracycline-free fetal bovine serum and induced with 4 µg ml -1 of doxycycline 4 days prior to treatment. Recombinant human interleukin-2 (rIL2) was obtained from the Biological Resources Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute."
},
{
"@type": "HowToStep",
"position": 8,
"name": "Tumour challenge and treatment",
"text": "Perpendicular tumour diameters were measured with a vernier caliper, and tumour volumes were calculated as length × width 2 × 0.52. When applicable, gene knockdown or knock-in expression were induced by adding doxycycline at 100 µg ml -1 (20 mg kg -1 day -1 ) into the mice drinking water at day 8, after tumours establishment. Doxycycline was replenished every 4 days until day 26. On day 12, when primary tumours reached an average size of 60-80 mm 3, animals were randomly assigned to different treatment groups. Radiotherapy (RT) was delivered to the tumour as previously described. Briefly, all mice (including mice receiving sham radiation) were anaesthetized by intra-peritoneal (i.p.) injection of avertin (240 mg kg -1 ) and the primary tumours irradiated with a single fraction of 8 Gy, 20 G..."
}
],
"tool": [
{
"@type": "HowToTool",
"name": "Patient-derived tumour xenograft"
},
{
"@type": "HowToTool",
"name": "In vivo fluorescent imaging"
},
{
"@type": "HowToTool",
"name": "Genome-wide microarray analysis"
},
{
"@type": "HowToTool",
"name": "Quantification of cytoplasmic dsDNA"
},
{
"@type": "HowToTool",
"name": "Genome-wide microarray analysis"
},
{
"@type": "HowToTool",
"name": "Gene expression analysis"
},
{
"@type": "HowToTool",
"name": "Flow cytometry analysis"
},
{
"@type": "HowToTool",
"name": "Western blot"
}
],
"supply": [
{
"@type": "HowToSupply",
"name": "Cells and reagents"
},
{
"@type": "HowToSupply",
"name": "Cells and reagents"
},
{
"@type": "HowToSupply",
"name": "In vivo fluorescent imaging"
},
{
"@type": "HowToSupply",
"name": "Genome-wide microarray analysis"
},
{
"@type": "HowToSupply",
"name": "Quantification of cytoplasmic dsDNA"
},
{
"@type": "HowToSupply",
"name": "Gene expression analysis"
},
{
"@type": "HowToSupply",
"name": "Gene expression analysis"
},
{
"@type": "HowToSupply",
"name": "Analysis of IFN-γ production by CD8 + T cells"
}
],
"isBasedOn": {
"@type": "ScholarlyArticle",
"headline": "DNA exonuclease Trex1 regulates radiotherapy-induced tumour immunogenicity",
"datePublished": "2017",
"author": [
{
"@type": "Person",
"name": "Claire Vanpouille-Box"
},
{
"@type": "Person",
"name": "Amandine Alard"
},
{
"@type": "Person",
"name": "Molykutty J. Aryankalayil"
},
{
"@type": "Person",
"name": "Yasmeen Sarfraz"
},
{
"@type": "Person",
"name": "Julie M. Diamond"
},
{
"@type": "Person",
"name": "Robert J. Schneider"
},
{
"@type": "Person",
"name": "Giorgio Inghirami"
},
{
"@type": "Person",
"name": "C. Norman Coleman"
},
{
"@type": "Person",
"name": "Silvia C. Formenti"
},
{
"@type": "Person",
"name": "Sandra Demaria"
}
],
"identifier": "10.1038/ncomms15618"
}
},
{
"@context": "https://schema.org",
"@type": "BreadcrumbList",
"itemListElement": [
{
"@type": "ListItem",
"position": 1,
"name": "Experiments",
"item": "https://replicatescience.com/experiments"
},
{
"@type": "ListItem",
"position": 2,
"name": "DNA exonuclease Trex1 regulates radiotherapy-induced tumour immunogenicity methods",
"item": "https://replicatescience.com/experiments/dna-exonuclease-trex1-regulates-radiotherapy-induced-tumour-immunogenicity-methods-claire-vanpouille-box-pmc5472757/dna-exonuclease-trex1-regulates-radiotherapy-induced-tumour-immunogenicity-mlpgvyag"
}
]
}
]