Chemotherapy-induced COX-2 upregulation by cancer cells defines their inflammatory properties and limits the efficacy of chemoimmunotherapy combinations

Cytotoxic therapies, besides directly inducing cancer cell death, can stimulate immune-dependent tumor growth control or paradoxically accelerate tumor progression. The underlying mechanisms dictating these opposing outcomes are poorly defined. Here, we show that cytotoxic therapy acutely upregulates cyclooxygenase (COX)-2 expression and prostaglandin E2 (PGE2) production in cancer cells with pre-existing COX-2 activity. Screening a compound library of 1280 approved drugs, we find that all classes of chemotherapy drugs enhance COX-2 transcription whilst arresting cancer cell proliferation. Genetic manipulation of COX-2 expression or its gene promoter region uncover how augmented COX-2/PGE2 activity post-treatment profoundly alters the inflammatory properties of chemotherapy-treated cancer cells in vivo. Pharmacological COX-2 inhibition boosts the efficacy of the combination of chemotherapy and PD-1 blockade. Crucially, in a poorly immunogenic breast cancer model, only the triple therapy unleashes tumor growth control and significantly reduces relapse and spontaneous metastatic spread in an adjuvant setting. Our findings suggest COX-2/PGE2 upregulation by dying cancer cells acts as a major barrier to cytotoxic therapy-driven tumor immunity and uncover a strategy to improve the outcomes of immunotherapy and chemotherapy combinations.


March 2021
Data Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A description of any restrictions on data availability -For clinical datasets or third party data, please ensure that the statement adheres to our policy Field-specific reporting Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection. The NCI-60 human cancer cell publically available data used in this study are available in the NCBI Gene Expression Omnibus database under accession code GSE116436. Source data are provided with this paper. The relevant data supporting the findings in this study are available in the Article, Supplementary  Stratified randomization was applied in order to normalize tumor sizes and body weights across treatment groups for in vivo experiments. All mice were assigned randomly into groups for peritoneal lavage experiments.
The investigators were not blinded to allocation of animals during experiments and outcome assessments. The counting of macroscopic lung metastases was performed by an investigator blinded to treatment group. The remaining experiments were not blinded as the investigators who set up the experiment analyzed the data, which is incompatible with blinding. Note that full information on the approval of the study protocol must also be provided in the manuscript.

Flow Cytometry
Plots Confirm that: The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
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None of the cell lines used were authenticated All cell lines were routinely tested and confirmed to be negative for mycoplasma No commonly misidentified cell lines were used All mice were maintained under pathogen-free conditions in ventilated cages with environment enrichment in the Biological Resources Unit at the CRUK Manchester Institute (CRUK MI), and allowed free access to irradiated food and autoclaved water ad libitum in a 12h light/dark cycle, with room temperature at 21 ± 2°C and a humidity of 45-65%. 6-12 week old female BALB/c mice (Envigo) and 12 week old female NSG mice (Charles River) were used in experiments. Mice were age-matched in experiments.

No wild animals were used
There are no field-collected samples All procedures involving animals were performed in accordance with the PDCC31AAF license approved by the Animal Welfare and Ethical Review Bodies (AWERB) of the CRUK Manchester Institute, and in accordance with National Home Office regulations under the Animals (Scientific Procedures) Act 1986 For analysis of peripheral blood leukocytes, 50µl peripheral blood was taken from mice via tail-vein into an EDTA-coated capillary and then 1.5ml tubes on ice. Samples were centrifuged at 300 rcf for 6 min at 4"C to separate plasma and cells, FACS buffer was added to the cell pellet and cell suspensions were moved to a 96-well V-bottom plate for antibody staining. Red blood cells were lysed using ACK buffer for 1min (Gibco).
For analysis of tumor-infiltrating leukocytes, tumors were collected into complete RPMI on ice. The surface of tumor samples were dried with paper and weights recorded. Samples were transferred into C-tubes (Miltenyi Biotech) containing RPMI and Collagenase IV (200 U/ml, Worthington Biochemical) and DNase I (0.2 mg/ml, Roche), then minced using scissors. The Ctubes were placed in a GentleMACS Octo Dissociator (Miltenyi Biotech), and tumors disaggregated with 2 rounds of the automated program m_impTumor_02_01. Dissociated tumors were incubated for 30 min at 37°C and disaggregated for one more round. The C-tubes were centrifuged and pellets resuspended in cold complete RPMI before being filtered through a 70µM cell strainer and pelleted. Cell suspensions were resuspended in FACS buffer. Fc receptors were saturated with anti-CD16/32 (clone 93, eBioscience) 5 min before staining. Cell viability was determined by Aqua LIVE/Dead-405nm staining