Fractionated but not single dose radiotherapy induces an immune-mediated abscopal effect when combined with anti-CTLA-4 antibody methods
Aim. Evidence-backed execution summary for Fractionated but not single dose radiotherapy induces an immune-mediated abscopal effect when combined with anti-CTLA-4 antibody methods from Fractionated but not single dose radiotherapy induces an immune-mediated abscopal effect when combined with anti-CTLA-4 antibody.
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
Purpose
reagent used in the protocol.
- Use
- This study tested the hypothesis that the type of dose-fractionation regimen determines the ability of radiotherapy to synergize with anti-CTLA-4 antibody.
Cell line and reagents
reagent used in the protocol.
- Use
- TSA is a BALB/c mouse-derived poorly immunogenic mammary carcinoma cell line ( ) and MCA38 is a C57BL/6 mouse-derived poorly immunogenic colon carcinoma ( ). TSA and MCA38 cells were cultured in DMEM medium (Invitrogen Corporation, Carlsbad, CA) supplemented with 2mM L-glutamine, 100 U/ml penicillin, 100 µg/ml...
Fractionated radiotherapy synergizes with anti-CTLA-4 antibody in the MCA38 colon cancer model
reagent used in the protocol.
- Use
- Administration of 9H10 as single modality did not enhance significantly CD4+ and CD8+ TIL in secondary MCA38 tumors, whereas TIL were markedly increased in mice treated with 8 Gy × 3 + 9H10 (p<0.05 for both CD4+ and CD8+ T cells, compared to control and 9H10 alone) ( ). The frequency of CD8+ T cells showing tum...
Immunostaining of tumor sections
reagent used in the protocol.
- Use
- Tumors from treated and untreated mice were harvested at Day 35 post tumor inoculation, fixed for 1 h at 4°C in 4% paraformaldehyde followed by overnight incubation in 30% sucrose, and frozen in optimum cutting temperature (OCT) medium. Sections (8 µm) were incubated with 0.1% Tween-20 and 0.01% Triton-X10...
Ex vivo production of IFN-γ by spleen cells
reagent used in the protocol.
- Use
- Spleen cells (1 × 10 6 ) from TSA tumor-bearing mice were cultured in 24-well tissue culture plates with 2.5 × 10 5 irradiated (20 Gy) TSA cells for 24 hrs in 1-ml fresh RPMI 1640 medium supplemented with 2 mM L-glutamine, 100 U/ml penicillin,100 µg/ml streptomycin, 50 µM 2-mercapthoethanol, 10% FBS...
Flow cytometry analysis of IFN-γ producing CD8 T cells
reagent used in the protocol.
- Use
- For in vitro re-stimulation, 3.5 × 10 6 spleen cells from TSA tumor-bearing mice were cultured with the TSA-derived immunodominant CD8 epitope AH1 peptide [SPSYVYHQF] (1 µM) ( ), whereas spleen cells from MCA38 tumor-bearing mice were cultured with 1 × 10 6 irradiated (50 Gy) MCA38 cells. After 5 days cul...
Effect of anti-CTLA-4 antibody administration schedule on TSA tumor inhibition in mice treated with radiotherapy
reagent used in the protocol.
- Use
- To determine whether the time of administration of 9H10 mAb relative to radiotherapy could play a role in its ability to induce an abscopal effect, 9H10 treatment was started on different days. As single modality, administration of 9H10 starting on day 12 did not show any effect on TSA tumor growth, similarly to wha...
Effect of anti-CTLA-4 antibody administration schedule on TSA tumor inhibition in mice treated with radiotherapy
reagent used in the protocol.
- Use
- Next, we tested whether starting 9H10 mAb treatment two days before the conclusion of fractionated radiotherapy (day 12), at conclusion (day 14) or two days later (day 16) affected the inhibition of tumor growth observed in mice treated with three fractions of 8 Gy. Primary tumor inhibition was significantly enhance...
Immunostaining of tumor sections
Tumors from treated and untreated mice were harvested at Day 35 post tumor inoculation, fixed for 1 h at 4°C in 4% paraformaldehyde followed by overnight incubation in 30% sucrose, and frozen in optimum cutting temperature (OCT) medium. Sections (8 µm) were incubated with 0.1% Tween-20 and 0.01% Triton-X10...
- Use
- Tumors from treated and untreated mice were harvested at Day 35 post tumor inoculation, fixed for 1 h at 4°C in 4% paraformaldehyde followed by overnight incubation in 30% sucrose, and frozen in optimum cutting temperature (OCT) medium. Sections (8 µm) were incubated with 0.1% Tween-20 and 0.01% Triton-X10...
Flow cytometry analysis of IFN-γ producing CD8 T cells
For in vitro re-stimulation, 3.5 × 10 6 spleen cells from TSA tumor-bearing mice were cultured with the TSA-derived immunodominant CD8 epitope AH1 peptide [SPSYVYHQF] (1 µM) ( ), whereas spleen cells from MCA38 tumor-bearing mice were cultured with 1 × 10 6 irradiated (50 Gy) MCA38 cells. After 5 days cul...
- Use
- For in vitro re-stimulation, 3.5 × 10 6 spleen cells from TSA tumor-bearing mice were cultured with the TSA-derived immunodominant CD8 epitope AH1 peptide [SPSYVYHQF] (1 µM) ( ), whereas spleen cells from MCA38 tumor-bearing mice were cultured with 1 × 10 6 irradiated (50 Gy) MCA38 cells. After 5 days cul...
Figures
(A, B) Secondary tumors were excised at day 35 and analyzed by fluorescence microscopy for the presence of CD4+ and CD8+ T cells. (A) Representative fields showing CD4+ (top panels) and CD8+ (bottom panels) T cells (white) infiltrating secondary TSA tumors in mice treated as indicated. Nuclei were stained with DAPI...
- Use
- (A, B) Secondary tumors were excised at day 35 and analyzed by fluorescence microscopy for the presence of CD4+ and CD8+ T cells. (A) Representative fields showing CD4+ (top panels) and CD8+ (bottom panels) T cells (white) infiltrating secondary TSA tumors in mice treated as indicated. Nuclei were stained with DAPI...
Figures
C57BL/6 mice were injected with syngeneic MCA38 colon carcinoma cells (5 × 10 5 ) s.c. into the right and left flank as outlined in. (A) Tumor growth delay of primary irradiated tumors (left panel) and secondary non-irradiated tumor (right panel) in mice treated with PBS (closed circles), 9H10 (open circles),...
- Use
- C57BL/6 mice were injected with syngeneic MCA38 colon carcinoma cells (5 × 10 5 ) s.c. into the right and left flank as outlined in. (A) Tumor growth delay of primary irradiated tumors (left panel) and secondary non-irradiated tumor (right panel) in mice treated with PBS (closed circles), 9H10 (open circles),...
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?
Purpose
This study tested the hypothesis that the type of dose-fractionation regimen determines the ability of radiotherapy to synergize with anti-CTLA-4 antibody.
Cell line and reagents
TSA is a BALB/c mouse-derived poorly immunogenic mammary carcinoma cell line ( ) and MCA38 is a C57BL/6 mouse-derived poorly immunogenic colon carcinoma ( ). TSA and MCA38 cells were cultured in DMEM medium (Invitrogen Corporation, Carlsbad, CA) supplemented with 2mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 2.5 × 10 -5 M 2-mercapthoethanol, and 10% FBS (Gemini Bio-Products Woodland, CA) (complete medium). These cells were found to be free of contamination by Mycoplasma by the Mycoplasma detection kit (Roche Diagnostics, Chicago, IL). Anti-CTLA-4 hamster mAb 9H10 was purified as previously described ( ).
Tumor challenge and treatment
BALB/c and C57BL/6 mice were injected s.c. with 1 × 10 5 TSA and 5 × 10 5 MCA38 cells, respectively, in the right flank on Day 0 (primary tumor) and in the left flank on Day 2 (secondary tumor). Perpendicular tumor diameters were measured with a Vernier caliper, and tumor volumes were calculated as length × width 2 × 0.52. On Day 12, when both tumors were palpable (average volume for TSA: 32 mm 3 and 21 mm 3 for primary ands secondary tumors, respectively; average volume for MCA38: 50 mm 3 and 25 mm 3 for primary and secondary tumors, respectively) animals were randomly assigned to various treatment groups, as indicated. Radiotherapy was administered as previously described ( ), with some modifications. Briefly, all mice (including mice receiving mock radiation) were lightly anesthetized by i.p. injection of Avertin (240mg/kg), positioned on a dedicated plexiglass tray...
Immunostaining of tumor sections
Tumors from treated and untreated mice were harvested at Day 35 post tumor inoculation, fixed for 1 h at 4°C in 4% paraformaldehyde followed by overnight incubation in 30% sucrose, and frozen in optimum cutting temperature (OCT) medium. Sections (8 µm) were incubated with 0.1% Tween-20 and 0.01% Triton-X100 for 20 minutes, followed by 4% rat serum in 4% BSA/PBS for an additional 30 minutes. Sections were stained with PE-Texas-Red-conjugated rat anti-mouse CD4 or PE-conjugated rat anti-mouse CD8α (Caltag, Carlsbad, CA), and counterstained with 5 µg/ml DAPI (Sigma). Images were obtained using a Nikon Eclipse 800 deconvolution microscope. Number of CD4 and CD8 T cells were counted in three randomly selected (20X) fields in each tumor.
Ex vivo production of IFN-γ by spleen cells
Spleen cells (1 × 10 6 ) from TSA tumor-bearing mice were cultured in 24-well tissue culture plates with 2.5 × 10 5 irradiated (20 Gy) TSA cells for 24 hrs in 1-ml fresh RPMI 1640 medium supplemented with 2 mM L-glutamine, 100 U/ml penicillin,100 µg/ml streptomycin, 50 µM 2-mercapthoethanol, 10% FBS (T cell medium). The supernatants were collected and stored at -80°C. IFNγ was measured in cell-free supernatants of duplicate wells by ELISA (Diaclone Tepnel, Lifecodes Corp. Stamford, CT). Tumor-specific IFNγ production was calculated by subtracting the background values measured in supernatants of spleen cells cultured with medium alone.
Flow cytometry analysis of IFN-γ producing CD8 T cells
For in vitro re-stimulation, 3.5 × 10 6 spleen cells from TSA tumor-bearing mice were cultured with the TSA-derived immunodominant CD8 epitope AH1 peptide [SPSYVYHQF] (1 µM) ( ), whereas spleen cells from MCA38 tumor-bearing mice were cultured with 1 × 10 6 irradiated (50 Gy) MCA38 cells. After 5 days culture in 24-well tissue culture plates in 2-ml T cell medium supplemented with 10 U/ml human rIL-2 (provided by the National Cancer Institute BRB Preclinical Repository) the percentage of CD8 T cells producing IFN-γ was determined. Briefly, T cells were cultured for 16 hrs with irradiated TSA or MCA38 target cell or with irrelevant target RMA-S Ld cells preloaded with MCMV peptide ( ) at 1:1 ratio in the presence of 1 µl/ml of Brefaldin A, washed and incubated with rat anti-mouse CD16/CD32 mAb (2.4G2) to block nonspecific binding and then stained with CD8α-PE-...
Effect of anti-CTLA-4 antibody administration schedule on TSA tumor inhibition in mice treated with radiotherapy
To determine whether the time of administration of 9H10 mAb relative to radiotherapy could play a role in its ability to induce an abscopal effect, 9H10 treatment was started on different days. As single modality, administration of 9H10 starting on day 12 did not show any effect on TSA tumor growth, similarly to what was observed when 9H10 was started on day 14 ( and ). Importantly, administration of 9H10 on day 12, 15 and 18 did not enhance the inhibition of either the primary or secondary tumors by a single 20 Gy dose yielding results similar to the delayed administration on day 14, 17 and 20 ( ). Consistent with these results, no significant interaction between 20 Gy and 9H10 started on day 12 was observed (p=0.21 and 0.42 for primary and secondary tumors, respectively). Therefore, the observed differential ability of single dose and fractionated radiotherapy to induce an abscopal...
Effect of anti-CTLA-4 antibody administration schedule on TSA tumor inhibition in mice treated with radiotherapy
Next, we tested whether starting 9H10 mAb treatment two days before the conclusion of fractionated radiotherapy (day 12), at conclusion (day 14) or two days later (day 16) affected the inhibition of tumor growth observed in mice treated with three fractions of 8 Gy. Primary tumor inhibition was significantly enhanced by administration of 9H10 on days 12, 15 and 18 or 14, 17 and 20 as compared to radiotherapy alone (p<0.001 for both schedules) ( ). Although 5 out of 6 primary tumors completely regressed with 3 fractions of 8 Gy plus 9H10 started at day 14, and only 3 out of 6 completely regressed when 9H10 was started at day 12, this difference was not statistically significant (p=0.08). Likewise, the growth of secondary tumors was significantly inhibited in both groups of mice (p<0.05 compared to control mice) and there was no significant difference when 9H10 mAb administration was st...
Measurement outputs
What raw and processed outputs should exist?
Administration of 9H10 as single modality did not enhance significantly CD4+ and CD8+ TIL in secondary MCA38 tumors, whereas TIL were markedly increased in mice treated with 8 G...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
Spleen cells (1 × 10 6 ) from TSA tumor-bearing mice were cultured in 24-well tissue culture plates with 2.5 × 10 5 irradiated (20 Gy) TSA cells for 24 hrs in 1-ml fresh R...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
For in vitro re-stimulation, 3.5 × 10 6 spleen cells from TSA tumor-bearing mice were cultured with the TSA-derived immunodominant CD8 epitope AH1 peptide [SPSYVYHQF] (1 _...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
Random coefficients regression was used to model log tumor volume and log tumor weight as functions of elapsed time from treatment onset and to compare treatment regimens with r...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
Analysis plan
How should the outputs become interpretable results?
Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
inferred from protocolPreprocessing / cleaning
Administration of 9H10 as single modality did not enhance significantly CD4+ and CD8+ TIL in secondary MCA38 tumors, whereas TIL were markedly increased in mice treated with 8 Gy × 3 + 9H10 (p<0.05 for both CD4+ and CD8+ T cells, compared to control and 9H10 alone) ( ).
from paperScoring or quantification
Quantify the primary readouts for this experiment: Administration of 9H10 as single modality did not enhance significantly CD4+ and CD8+ TIL in secondary MCA38 tumors, whereas TIL were markedly increased in mice treated with 8 G...; Spleen cells (1 × 10 6 ) from TSA tumor-bearing mice were cultured in 24-well tissue culture plates with 2.5 × 10 5 irradiated (20 Gy) TSA cells for 24 hrs in 1-ml fresh R...; For in vitro re-stimulation, 3.5 × 10 6 spleen cells from TSA tumor-bearing mice were cultured with the TSA-derived immunodominant CD8 epitope AH1 peptide [SPSYVYHQF] (1 _...; Random coefficients regression was used to model log tumor volume and log tumor weight as functions of elapsed time from treatment onset and to compare treatment regimens with r....
from paperStatistical comparison
Administration of 9H10 as single modality did not enhance significantly CD4+ and CD8+ TIL in secondary MCA38 tumors, whereas TIL were markedly increased in mice treated with 8 G...; Random coefficients regression was used to model log tumor volume and log tumor weight as functions of elapsed time from treatment onset and to compare treatment regimens with r...; To determine whether the time of administration of 9H10 mAb relative to radiotherapy could play a role in its ability to induce an abscopal effect, 9H10 treatment was started on...; Next, we tested whether starting 9H10 mAb treatment two days before the conclusion of fractionated radiotherapy (day 12), at conclusion (day 14) or two days later (day 16) affec...
from paperReporting output
Report representative outputs alongside summary comparisons for Administration of 9H10 as single modality did not enhance significantly CD4+ and CD8+ TIL in secondary MCA38 tumors, whereas TIL were markedly increased in mice treated with 8 G..., Spleen cells (1 × 10 6 ) from TSA tumor-bearing mice were cultured in 24-well tissue culture plates with 2.5 × 10 5 irradiated (20 Gy) TSA cells for 24 hrs in 1-ml fresh R..., For in vitro re-stimulation, 3.5 × 10 6 spleen cells from TSA tumor-bearing mice were cultured with the TSA-derived immunodominant CD8 epitope AH1 peptide [SPSYVYHQF] (1 _..., Random coefficients regression was used to model log tumor volume and log tumor weight as functions of elapsed time from treatment onset and to compare treatment regimens with r....
inferred from protocolStructured statistical methods
Administration of 9H10 as single modality did not enhance significantly CD4+ and CD8+ TIL in secondary MCA38 tumors, whereas TIL were markedly increased in mice treated with 8 G...; Random coefficients regression was used to model log tumor volume and log tumor weight as functions of elapsed time from treatment onset and to compare treatment regimens with r...; To determine whether the time of administration of 9H10 mAb relative to radiotherapy could play a role in its ability to induce an abscopal effect, 9H10 treatment was started on...; Next, we tested whether starting 9H10 mAb treatment two days before the conclusion of fractionated radiotherapy (day 12), at conclusion (day 14) or two days later (day 16) affec...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (8)
This study tested the hypothesis that the type of dose-fractionation regimen determines the ability of radiotherapy to synergize with anti-CTLA-4 antibody.
TSA is a BALB/c mouse-derived poorly immunogenic mammary carcinoma cell line ( ) and MCA38 is a C57BL/6 mouse-derived poorly immunogenic colon carcinoma ( ). TSA and MCA38 cells were cultured in DMEM medium (Invitrogen Corporation, Carlsbad, CA) supplemented with 2mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 2.5 × 10 -5 M 2-mercapthoethanol, and 10% FBS (Gemini Bio-Products Woodland, CA) (complete medium). These cells were found to be free of contamination by Mycoplasma by the Mycoplasma detection kit (Roche Diagnostics, Chicago, IL). Anti-CTLA-4 hamster mAb 9H10 was purified as previously described ( ).
BALB/c and C57BL/6 mice were injected s.c. with 1 × 10 5 TSA and 5 × 10 5 MCA38 cells, respectively, in the right flank on Day 0 (primary tumor) and in the left flank on Day 2 (secondary tumor). Perpendicular tumor diameters were measured with a Vernier caliper, and tumor volumes were calculated as length × width 2 × 0.52. On Day 12, when both tumors were palpable (average volume for TSA: 32 mm 3 and 21 mm 3 for primary ands secondary tumors, respectively; average volume for MCA38: 50 mm 3 and 25 mm 3 for primary and secondary tumors, respectively) animals were randomly assigned to various treatment groups, as indicated. Radiotherapy was administered as previously described ( ), with some modifications. Briefly, all mice (including mice receiving mock radiation) were lightly anesthetized by i.p. injection of Avertin (240mg/kg), positioned on a dedicated plexiglass tray and the whole body was protected by lead shielding, except for the area of the tumor to be irradiated. Radiotherapy was delivered to a field including the tumor with 5 mm margins using a Clinac 2300 C/D Linear Accelerator (Varian Medical Systems, Palo Alto, Ca.) fitted with a 25 mm RadioSurgery con...
Tumors from treated and untreated mice were harvested at Day 35 post tumor inoculation, fixed for 1 h at 4°C in 4% paraformaldehyde followed by overnight incubation in 30% sucrose, and frozen in optimum cutting temperature (OCT) medium. Sections (8 µm) were incubated with 0.1% Tween-20 and 0.01% Triton-X100 for 20 minutes, followed by 4% rat serum in 4% BSA/PBS for an additional 30 minutes. Sections were stained with PE-Texas-Red-conjugated rat anti-mouse CD4 or PE-conjugated rat anti-mouse CD8α (Caltag, Carlsbad, CA), and counterstained with 5 µg/ml DAPI (Sigma). Images were obtained using a Nikon Eclipse 800 deconvolution microscope. Number of CD4 and CD8 T cells were counted in three randomly selected (20X) fields in each tumor.
Spleen cells (1 × 10 6 ) from TSA tumor-bearing mice were cultured in 24-well tissue culture plates with 2.5 × 10 5 irradiated (20 Gy) TSA cells for 24 hrs in 1-ml fresh RPMI 1640 medium supplemented with 2 mM L-glutamine, 100 U/ml penicillin,100 µg/ml streptomycin, 50 µM 2-mercapthoethanol, 10% FBS (T cell medium). The supernatants were collected and stored at -80°C. IFNγ was measured in cell-free supernatants of duplicate wells by ELISA (Diaclone Tepnel, Lifecodes Corp. Stamford, CT). Tumor-specific IFNγ production was calculated by subtracting the background values measured in supernatants of spleen cells cultured with medium alone.
For in vitro re-stimulation, 3.5 × 10 6 spleen cells from TSA tumor-bearing mice were cultured with the TSA-derived immunodominant CD8 epitope AH1 peptide [SPSYVYHQF] (1 µM) ( ), whereas spleen cells from MCA38 tumor-bearing mice were cultured with 1 × 10 6 irradiated (50 Gy) MCA38 cells. After 5 days culture in 24-well tissue culture plates in 2-ml T cell medium supplemented with 10 U/ml human rIL-2 (provided by the National Cancer Institute BRB Preclinical Repository) the percentage of CD8 T cells producing IFN-γ was determined. Briefly, T cells were cultured for 16 hrs with irradiated TSA or MCA38 target cell or with irrelevant target RMA-S Ld cells preloaded with MCMV peptide ( ) at 1:1 ratio in the presence of 1 µl/ml of Brefaldin A, washed and incubated with rat anti-mouse CD16/CD32 mAb (2.4G2) to block nonspecific binding and then stained with CD8α-PE-Cy5 and IFN-γ-FITC or control antibodies according to the manufacture's instructions (BD PharMingen). Cells were analyzed using a FACScan flow cytometer and FlowJo version 8.7.1 (Tree Star, Ashland, OR).
To determine whether the time of administration of 9H10 mAb relative to radiotherapy could play a role in its ability to induce an abscopal effect, 9H10 treatment was started on different days. As single modality, administration of 9H10 starting on day 12 did not show any effect on TSA tumor growth, similarly to what was observed when 9H10 was started on day 14 ( and ). Importantly, administration of 9H10 on day 12, 15 and 18 did not enhance the inhibition of either the primary or secondary tumors by a single 20 Gy dose yielding results similar to the delayed administration on day 14, 17 and 20 ( ). Consistent with these results, no significant interaction between 20 Gy and 9H10 started on day 12 was observed (p=0.21 and 0.42 for primary and secondary tumors, respectively). Therefore, the observed differential ability of single dose and fractionated radiotherapy to induce an abscopal effect ( ) can not be explained by the two days interval between the 20 Gy irradiation and the beginning of immunotherapy.
Next, we tested whether starting 9H10 mAb treatment two days before the conclusion of fractionated radiotherapy (day 12), at conclusion (day 14) or two days later (day 16) affected the inhibition of tumor growth observed in mice treated with three fractions of 8 Gy. Primary tumor inhibition was significantly enhanced by administration of 9H10 on days 12, 15 and 18 or 14, 17 and 20 as compared to radiotherapy alone (p<0.001 for both schedules) ( ). Although 5 out of 6 primary tumors completely regressed with 3 fractions of 8 Gy plus 9H10 started at day 14, and only 3 out of 6 completely regressed when 9H10 was started at day 12, this difference was not statistically significant (p=0.08). Likewise, the growth of secondary tumors was significantly inhibited in both groups of mice (p<0.05 compared to control mice) and there was no significant difference when 9H10 mAb administration was started on day 12 or 14 (p=0.9) ( ). Delaying administration of 9H10 mAb until day 16 reduced the therapeutic effect, with only 1 out of 6 primary tumors showing complete regression, and a reduced growth inhibition of the secondary tumors ( ). This suggests that delaying immunotherapy may reduce its p...
Machine-readable layer
[
{
"@context": "https://schema.org",
"@type": "HowTo",
"name": "Fractionated but not single dose radiotherapy induces an immune-mediated abscopal effect when combined with anti-CTLA-4 antibody methods",
"description": "Evidence-backed execution summary for Fractionated but not single dose radiotherapy induces an immune-mediated abscopal effect when combined with anti-CTLA-4 antibody methods from Fractionated but not single dose radiotherapy induces an immune-mediated abscopal effect when combined with anti-CTLA-4 antibody.",
"totalTime": "PT3860M",
"step": [
{
"@type": "HowToStep",
"position": 1,
"name": "Purpose",
"text": "This study tested the hypothesis that the type of dose-fractionation regimen determines the ability of radiotherapy to synergize with anti-CTLA-4 antibody."
},
{
"@type": "HowToStep",
"position": 2,
"name": "Cell line and reagents",
"text": "TSA is a BALB/c mouse-derived poorly immunogenic mammary carcinoma cell line ( ) and MCA38 is a C57BL/6 mouse-derived poorly immunogenic colon carcinoma ( ). TSA and MCA38 cells were cultured in DMEM medium (Invitrogen Corporation, Carlsbad, CA) supplemented with 2mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 2.5 × 10 -5 M 2-mercapthoethanol, and 10% FBS (Gemini Bio-Products Woodland, CA) (complete medium). These cells were found to be free of contamination by Mycoplasma by the Mycoplasma detection kit (Roche Diagnostics, Chicago, IL). Anti-CTLA-4 hamster mAb 9H10 was purified as previously described ( )."
},
{
"@type": "HowToStep",
"position": 3,
"name": "Tumor challenge and treatment",
"text": "BALB/c and C57BL/6 mice were injected s.c. with 1 × 10 5 TSA and 5 × 10 5 MCA38 cells, respectively, in the right flank on Day 0 (primary tumor) and in the left flank on Day 2 (secondary tumor). Perpendicular tumor diameters were measured with a Vernier caliper, and tumor volumes were calculated as length × width 2 × 0.52. On Day 12, when both tumors were palpable (average volume for TSA: 32 mm 3 and 21 mm 3 for primary ands secondary tumors, respectively; average volume for MCA38: 50 mm 3 and 25 mm 3 for primary and secondary tumors, respectively) animals were randomly assigned to various treatment groups, as indicated. Radiotherapy was administered as previously described ( ), with some modifications. Briefly, all mice (including mice receiving mock radiation) were lightly anesthetized by i.p. injection of Avertin (240mg/kg), positioned on a dedicated plexiglass tray..."
},
{
"@type": "HowToStep",
"position": 4,
"name": "Immunostaining of tumor sections",
"text": "Tumors from treated and untreated mice were harvested at Day 35 post tumor inoculation, fixed for 1 h at 4°C in 4% paraformaldehyde followed by overnight incubation in 30% sucrose, and frozen in optimum cutting temperature (OCT) medium. Sections (8 µm) were incubated with 0.1% Tween-20 and 0.01% Triton-X100 for 20 minutes, followed by 4% rat serum in 4% BSA/PBS for an additional 30 minutes. Sections were stained with PE-Texas-Red-conjugated rat anti-mouse CD4 or PE-conjugated rat anti-mouse CD8α (Caltag, Carlsbad, CA), and counterstained with 5 µg/ml DAPI (Sigma). Images were obtained using a Nikon Eclipse 800 deconvolution microscope. Number of CD4 and CD8 T cells were counted in three randomly selected (20X) fields in each tumor."
},
{
"@type": "HowToStep",
"position": 5,
"name": "Ex vivo production of IFN-γ by spleen cells",
"text": "Spleen cells (1 × 10 6 ) from TSA tumor-bearing mice were cultured in 24-well tissue culture plates with 2.5 × 10 5 irradiated (20 Gy) TSA cells for 24 hrs in 1-ml fresh RPMI 1640 medium supplemented with 2 mM L-glutamine, 100 U/ml penicillin,100 µg/ml streptomycin, 50 µM 2-mercapthoethanol, 10% FBS (T cell medium). The supernatants were collected and stored at -80°C. IFNγ was measured in cell-free supernatants of duplicate wells by ELISA (Diaclone Tepnel, Lifecodes Corp. Stamford, CT). Tumor-specific IFNγ production was calculated by subtracting the background values measured in supernatants of spleen cells cultured with medium alone."
},
{
"@type": "HowToStep",
"position": 6,
"name": "Flow cytometry analysis of IFN-γ producing CD8 T cells",
"text": "For in vitro re-stimulation, 3.5 × 10 6 spleen cells from TSA tumor-bearing mice were cultured with the TSA-derived immunodominant CD8 epitope AH1 peptide [SPSYVYHQF] (1 µM) ( ), whereas spleen cells from MCA38 tumor-bearing mice were cultured with 1 × 10 6 irradiated (50 Gy) MCA38 cells. After 5 days culture in 24-well tissue culture plates in 2-ml T cell medium supplemented with 10 U/ml human rIL-2 (provided by the National Cancer Institute BRB Preclinical Repository) the percentage of CD8 T cells producing IFN-γ was determined. Briefly, T cells were cultured for 16 hrs with irradiated TSA or MCA38 target cell or with irrelevant target RMA-S Ld cells preloaded with MCMV peptide ( ) at 1:1 ratio in the presence of 1 µl/ml of Brefaldin A, washed and incubated with rat anti-mouse CD16/CD32 mAb (2.4G2) to block nonspecific binding and then stained with CD8α-PE-..."
},
{
"@type": "HowToStep",
"position": 7,
"name": "Effect of anti-CTLA-4 antibody administration schedule on TSA tumor inhibition in mice treated with radiotherapy",
"text": "To determine whether the time of administration of 9H10 mAb relative to radiotherapy could play a role in its ability to induce an abscopal effect, 9H10 treatment was started on different days. As single modality, administration of 9H10 starting on day 12 did not show any effect on TSA tumor growth, similarly to what was observed when 9H10 was started on day 14 ( and ). Importantly, administration of 9H10 on day 12, 15 and 18 did not enhance the inhibition of either the primary or secondary tumors by a single 20 Gy dose yielding results similar to the delayed administration on day 14, 17 and 20 ( ). Consistent with these results, no significant interaction between 20 Gy and 9H10 started on day 12 was observed (p=0.21 and 0.42 for primary and secondary tumors, respectively). Therefore, the observed differential ability of single dose and fractionated radiotherapy to induce an abscopal..."
},
{
"@type": "HowToStep",
"position": 8,
"name": "Effect of anti-CTLA-4 antibody administration schedule on TSA tumor inhibition in mice treated with radiotherapy",
"text": "Next, we tested whether starting 9H10 mAb treatment two days before the conclusion of fractionated radiotherapy (day 12), at conclusion (day 14) or two days later (day 16) affected the inhibition of tumor growth observed in mice treated with three fractions of 8 Gy. Primary tumor inhibition was significantly enhanced by administration of 9H10 on days 12, 15 and 18 or 14, 17 and 20 as compared to radiotherapy alone (p<0.001 for both schedules) ( ). Although 5 out of 6 primary tumors completely regressed with 3 fractions of 8 Gy plus 9H10 started at day 14, and only 3 out of 6 completely regressed when 9H10 was started at day 12, this difference was not statistically significant (p=0.08). Likewise, the growth of secondary tumors was significantly inhibited in both groups of mice (p<0.05 compared to control mice) and there was no significant difference when 9H10 mAb administration was st..."
}
],
"tool": [
{
"@type": "HowToTool",
"name": "Immunostaining of tumor sections"
},
{
"@type": "HowToTool",
"name": "Flow cytometry analysis of IFN-γ producing CD8 T cells"
},
{
"@type": "HowToTool",
"name": "Figures"
},
{
"@type": "HowToTool",
"name": "Figures"
}
],
"supply": [
{
"@type": "HowToSupply",
"name": "Purpose"
},
{
"@type": "HowToSupply",
"name": "Cell line and reagents"
},
{
"@type": "HowToSupply",
"name": "Fractionated radiotherapy synergizes with anti-CTLA-4 antibody in the MCA38 colon cancer model"
},
{
"@type": "HowToSupply",
"name": "Immunostaining of tumor sections"
},
{
"@type": "HowToSupply",
"name": "Ex vivo production of IFN-γ by spleen cells"
},
{
"@type": "HowToSupply",
"name": "Flow cytometry analysis of IFN-γ producing CD8 T cells"
},
{
"@type": "HowToSupply",
"name": "Effect of anti-CTLA-4 antibody administration schedule on TSA tumor inhibition in mice treated with radiotherapy"
},
{
"@type": "HowToSupply",
"name": "Effect of anti-CTLA-4 antibody administration schedule on TSA tumor inhibition in mice treated with radiotherapy"
}
],
"isBasedOn": {
"@type": "ScholarlyArticle",
"headline": "Fractionated but not single dose radiotherapy induces an immune-mediated abscopal effect when combined with anti-CTLA-4 antibody",
"datePublished": "2009",
"author": [
{
"@type": "Person",
"name": "M. Zahidunnabi Dewan"
},
{
"@type": "Person",
"name": "Ashley E. Galloway"
},
{
"@type": "Person",
"name": "Noriko Kawashima"
},
{
"@type": "Person",
"name": "J. Keith Dewyngaert"
},
{
"@type": "Person",
"name": "James S. Babb"
},
{
"@type": "Person",
"name": "Silvia C. Formenti"
},
{
"@type": "Person",
"name": "Sandra Demaria"
}
],
"identifier": "10.1158/1078-0432.ccr-09-0265"
}
},
{
"@context": "https://schema.org",
"@type": "BreadcrumbList",
"itemListElement": [
{
"@type": "ListItem",
"position": 1,
"name": "Experiments",
"item": "https://replicatescience.com/experiments"
},
{
"@type": "ListItem",
"position": 2,
"name": "Fractionated but not single dose radiotherapy induces an immune-mediated abscopal effect when combined with anti-CTLA-4 antibody methods",
"item": "https://replicatescience.com/experiments/fractionated-but-not-single-dose-radiotherapy-induces-an-immune-mediated-abscopal-effect-when-combined-with-anti-ctla-4-antibody-methods-m-zahidunnabi-dewan-pmc2746048/fractionated-but-not-single-dose-radiotherapy-induces-an-immune-mediated-abscopal-effect-when-combin-mlpgvz5x"
}
]
}
]