NFκB in Pancreatic Stellate Cells Reduces Infiltration of Tumors by Cytotoxic T Cells and Killing of Cancer Cells, via Up-regulation of CXCL12

Abstract

Background & aims: Immunotherapies are ineffective against pancreatic cancer. We investigated whether the activity of nuclear factor (NF)κB in pancreatic stromal cells contributes to an environment that suppresses antitumor immune response.

Methods: Pancreata of C57BL/6 or Rag1^-/- mice were given pancreatic injections of a combination of Kras^G12D/+; Trp53 ^R172H/+; Pdx-1cre (KPC) pancreatic cancer cells and pancreatic stellate cells (PSCs) extracted from C57BL/6 (control) or mice with disruption of the gene encoding the NFκB p50 subunit (Nfkb1 or p50^-/- mice). Tumor growth was measured as an endpoint. Other mice were given injections of Lewis lung carcinoma (LLC) lung cancer cells or B16-F10 melanoma cells with control or p50^-/- fibroblasts. Cytotoxic T cells were depleted from C57BL/6 mice by administration of antibodies against CD8 (anti-CD8), and growth of tumors from KPC cells, with or without control or p50^-/- PSCs, was measured. Some mice were given an inhibitor of CXCL12 (AMD3100) and tumor growth was measured. T-cell migration toward cancer cells was measured using the Boyden chamber assay.

Results: C57BL/6 mice coinjected with KPC cells (or LLC or B16-F10 cells) and p50^-/- PSCs developed smaller tumors than mice given injections of the cancer cells along with control PSCs. Tumors that formed when KPC cells were injected along with p50^-/- PSCs had increased infiltration by activated cytotoxic T cells along with decreased levels of CXCL12, compared with tumors grown from KPC cells injected along with control PSCs. KPC cells, when coinjected with control or p50^-/- PSCs, developed the same-size tumors when CD8⁺ T cells were depleted from C57BL/6 mice or in Rag1^-/- mice. The CXCL12 inhibitor slowed tumor growth and increased tumor infiltration by cytotoxic T cells. In vitro expression of p50 by PSCs reduced T-cell migration toward and killing of cancer cells. When cultured with cancer cells, control PSCs expressed 10-fold higher levels of CXCL12 than p50^-/- PSCs. The CXCL12 inhibitor increased migration of T cells toward KPC cells in culture.

Conclusions: In studies of mice and cell lines, we found that NFκB activity in PSCs promotes tumor growth by increasing expression of CXCL12, which prevents cytotoxic T cells from infiltrating the tumor and killing cancer cells. Strategies to block CXCL12 in pancreatic tumor cells might increase antitumor immunity.

Keywords: CXCR4; Chemokine; Cytokine; Immune Suppression.