Tumor-driven stromal reprogramming in the pre-metastatic lymph node

Metastatic dissemination is critically reliant on the formation of a receptive niche, a process which is thought to rely on signals derived from the primary tumor. Lymph nodes are continuously exposed to such signals through the flow of afferent lymph, allowing the potential reprograming of lymphoid tissue stroma in support of metastases or immunosuppression. We utilize single cell RNA sequencing to profile reprograming of the stromal support network within the pre-metastatic lymph node. Transcriptomic changes of the tumor-draining LN are compared against those of the contralateral non-draining LN. Additionally, we include experimental conditions for comparison in which LNs are exposed to fixed tumor cells, mimicking antigenic challenge without active tumor growth, thereby allowing us to distinguish between active tumor-elicited reprograming and general immune responses to tumor antigen. Overall design: Single Cell RNA sequencing was performed on enriched PDPN+ stromal cells isolated from enzymatically dissociated lymph nodes. Tumor-draining and non-draining inguinal lymph nodes were harvested from mice bearing subcutaneous MC38 tumors. Draining and non-draining inguinal lymph nodes were also harvested from mice inoculated with fixed (non-viable) MC38 cancer cells. For both conditions, 5 x 105 cells were implanted subcutaneously on the animal's lower-right flank. Mice were euthanized and LNs were collected on day 12 after treatment.