DOI: 10.5061/dryad.n2z34tn5z
title: Data for: Cytosolic bacterial pathogens activate TLR pathways in tumors that synergistically enhance STING agonist cancer therapies

description: Bacterial pathogens that invade the eukaryotic cytosol are distinctive
 tools for fighting cancer, as they preferentially target tumors and can
 deliver cancer antigens to MHC-I. Cytosolic bacterial pathogens have
 undergone extensive preclinical development and human clinical trials, yet
 the molecular mechanisms by which they activate innate immunity in tumors
 are unclear. We report that intratumoral delivery of phylogenetically
 distinct cytosolic pathogens, including Listeria, Rickettsia, and
 Burkholderia species, elicited anti-tumor responses in established, poorly
 immunogenic melanoma and lymphoma in mice. We were surprised to observe
 that although the bacteria required entry to the cytosol, the anti-tumor
 responses were largely independent of the cytosolic sensors cGAS and STING
 and instead required TLR signaling. Combining pathogens with STING
 agonists elicited profound, synergistic anti-tumor effects with complete
 responses in &gt;80% of mice after a single dose. Small-molecule TLR
 agonists also synergistically enhanced the anti-tumor activity of STING
 agonists. The anti-tumor effects required RAG2 but not interferon
 signaling. Mice cured from the combination therapy developed immunity to
 cancer rechallenge that required CD8+ T cells. Together, these data
 provide a framework for enhancing the efficacy of microbial cancer
 therapies and small-molecule innate immune agonists, via the co-activation
 of STING and TLRs.
description: Tumors were injected on the day that they measured approximately
 6 x 6 x 2.5 mm in each direction. Tumor volumes are reported as ellipsoids
 using the formula: <em>V</em> = (π/6)<em>ABC</em>.
 For any days not measured, tumor volumes were averaged between days of
 measurement. If animals were euthanized, tumor volumes were measured until
 the last mouse from each group was euthanized.
description: # Data for: Cytosolic bacterial pathogens activate TLR pathways in tumors
 that synergistically enhance STING agonist cancer therapies
 [https://doi.org/10.5061/dryad.n2z34tn5z](https://doi.org/10.5061/dryad.n2z34tn5z) ## Description of the data and file structure Figures in this manuscript fall into 3 categories: 1) tumor volume, 2) mouse survival, and 3) type I interferon (IFN-I) production. **1) Tumor volume: Figures 1A, 1B, 1C, 1D, 1E, 2A, 2B, 3A-3D, 3F-3J, 4A, 4B, 4D, 4E, 5A-F, 6B, 7A** The way to interpret these data is that the Y axis is tumor volume, and the X axis is time. Each column of data is an individual mouse. Mice were injected with the anti-cancer agents on day 0, when tumor volumes measured approximately 6 x 6 x 2.5 mm in each direction. Tumor volumes are reported as ellipsoids using the formula: *V* = (π/6)*ABC*. For any days not measured, tumor volumes were averaged between days of measurement. If animals were euthanized, tumor volumes were measured until the last mouse from each group was euthanized. n/a signifies that the data are unavailable because the animal was euthanized. **2) Mouse survival: Figures 1A, 1B, 1C, 2A, 2B, 3E, 4C, 4F, 5G, 5H, 5I, 7B, 8A, 8B** Mice were euthanized when tumor volumes reached 15 mm in any one direction. These data report how long mice survived until their euthanasia. A 1 represents that the animal was euthanized, and a 0 means that the animal survived the entirety of the experiment to the day listed. n/a signifies that the data are unavailable because the animal was euthanized. **3) IFN-I production: Fig. 6A.** IFN-I is a pro-inflammatory cytokine produced downstream of innate immune sensors including STING. IFN-I was measured after infection or treatment of macrophages, then supernatants were collected and used to treat an IFN-I responsive cell line. 4 h later these cells were lysed and relative light units (RLU) were measured after adding a luciferin reagent. Data are expressed as fold change over uninfected. The titles for each Figure are below: Figure 1: Intratumoral delivery of cytosolic bacterial pathogens elicits dose-dependent anti-tumor responses in multiple non-immunogenic murine tumor models Figure 2: Cytosolic access of bacteria promotes the anti-tumor response Figure 3: The microbe-mediated anti-tumor effects are largely independent of cGAS/STING but require TLR signaling Figure 4: RAG2 is required for the microbe-mediated anti-tumor effects, while IFNAR or IFNGR are largely dispensable Figure 5: The anti-tumor effects of bacterial pathogens synergize with the STING agonist S100 Figure 6: IFN-I induction by STING agonists is maintained in infected cells *in vitro*, and IFNs are not critical for the anti-tumor response *in vivo* Figure 7: Small molecule STING+TLR agonist combinations elicit synergistic anti-tumor effects Figure 8: Bacterial cancer therapies elicit immunity to tumor cell rechallenge **Variable data and abbreviations:** ### Files and variables #### File: Cell\_Press\_Data\_Record\_For\_submission.xlsx **Description:** All tumor volume and survival data, Danielson et al. 2024 ##### Variables * All reported data of tumor volumes are in cubic millimeters (mm^3). * Each column is an individual mouse for tumor volume data. * All survival data is reported as a "1" for the day that an animal was euthanized or a "0" if the animal survived - Data that are "n/a" means that the mouse was euthanized and therefore no further tumor volumes were collected. - All drugs/bacteria were administered at day = 0, unless otherwise specified. - Tumor volume was measured over time and is expressed as an ellipsoid. - S100, a STING agonist - The number of bacteria delivered is indicated in log format, for example, "10^7" is 10,000,000. - Naive means that mice were not immunized. **Abbreviations**: Rp or *R. parkeri* - *Rickettsia parkeri* Lm or *L. monocytogenes* - *Listeria monocytogenes* Bt or *B. thailandensis* - *Burkholderia thailandends* PBS - phosphate-buffered saline CDN - cyclic dinucleotide (activator of the protein STING) PAM = PAM3CSK4, a TLR2 agonist IFNAR = type I interferon receptor IFNGR = type II / interferon gamma receptor cGAS = cyclic GMP AMP synthase Any other abbreviations will be denoted in the manuscript file. ## Code/software NA ## Access information NA