Data-driven learning how oncogenic gene expression locally alters heterocellular networks

Developing drugs increasingly relies on mechanistic modeling and simulation. Models that capture causal relations among genetic drivers of oncogenesis, functional plasticity, and host immunity complement wet experiments. Unfortunately, formulating such mechanistic cell-level models currently relies on hand curation, which can bias how data is interpreted or the priority of drug targets. In modeling molecular-level networks, rules and algorithms are employed to limit a priori biases in formulating mechanistic models. Here we combine digital cytometry with Bayesian network inference to generate causal models of cell-level networks linking an increase in gene expression associated with oncogenesis with alterations in stromal and immune cell subsets from bulk transcriptomic datasets. We predict how increased Cell Communication Network factor 4, a secreted matricellular protein, alters the tumor microenvironment using data from patients diagnosed with breast cancer and melanoma. Predictions are then tested using two immunocompetent mouse models for melanoma, which provide consistent experimental results.


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. Transcriptomics profiling of bulk tissue samples using Illumina RNA sequencing for the breast cancer (BRCA) and cutaneous melanoma (SKCM) arms of the Cancer Genome Atlas was downloaded from TCGA data commons, where values for gene expression were expressed in counts, using the "TCGAbiolinks" (V2.8.2) package in R (V3.6.1) and converted to TPM. This is detailed in the GitHub repository.
As this was an exploratory study, no sample-size calculation was performed prior to the study. However, independent experiments using multiple replicates are used to ensure reproducibility. The size of a cohort within a biological replicate was limited to the bandwidth of sample processing workflow.
Mice were excluded from the study if they failed to develop tumors following subcutaneous challenge.
Experiments were repeated at least once, or data were compiled from two independent experiments. Replicates were reproducible.
Mice were purchased from the indicated vendors, labeled and randomized to treatment groups/cages. Mice receiving different cell lines were housed in the same cages, at a density of five mice per cage.
The investigators were not blinded to the group allocation during data collection or analysis, as the same individuals that set up the experiments were the ones that analyzed the results. They did remain objective in interpreting the data.
In all assays, purified rat anti-mouse CD16/CD32 antibodies (Mouse BD Fc Block, BD Biosciences #553142) were used to block Fc receptors prior to antibody staining.
To analyze tumor-infiltrating T cells, the following anti-mouse antibodies and dyes were used: LIVE/DEAD Fix --Violet/Pacific Blue 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).
The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells or percentage (with statistics) is provided.
To analyze tumor-infiltrating myeloid cells, the following anti-mouse antibodies and dyes were used: LIVE/DEAD Fix --Violet/Pacific Blue (Invitrogen #L34963); CD45 BB515 ( All antibodies were purchased from the vendors mentioned above, where they are validated by the manufacturers and validation data are available at the manufacturers' websites. These antibodies are routinely used in our laboratory without additional validation. , who created the cell line. All cell lines were revived from frozen stock, used within 10-15 passages that did not exceed a period of 6 months.
Cell lines were not authenticated.
Cells were routinely tested for mycoplasma contamination by PCR, and found to be negative.
None of the cell lines used were listed in the ICLAC database.
C57BL/6Ncrl mice (6-8 week-old, female) were purchased from Charles River Laboratories. Animals were housed with a 12-hour light/ dark cycle (light 6 am to 6pm), temperature nominally 74 degrees F and humidity 50%. If temperature is out of the range of 68 to 79 or if the humidity is not in the range of 30 -70%, it will alarm.
The study did not involve wild animals.
The study did not involve samples collected from the field.
All animal experiments were approved by West Virginia University (WVU) Institutional Animal Care and Use Committee and performed at the WVU Animal Facility (IACUC Protocol #1604002138).