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.

Figure. 1. NFKB1 in stroma is required for the growth of cancer.

A. Lack of p50^−/− in stromal cells leads to reduced growth of KPC pancreatic tumors. KPC pancreatic cancer cells were either injected alone (KPC) or co-injected with WT-PSC (KPC+WT-PSC) or p50^−/−-PSC (KPC+p50^−/− PSC) mice in the pancreas of WT-mice and allowed to grow for 30 days (n=at least 8 animals per group). *p<0.05. B. Pancreatic tumor sections harvested from different groups were stained with H&E or Sirius red (Collagen). IHC was performed for α-SMA and Vimentin. Quantification from 10 different fields of examination for 5 different mice is depicted. *p<0.05. C. Lack of NFκB in tumor stroma increases survival of mice with KPC pancreatic cancer. Mice with KPC, KPC+WT-PSC and KPC+p50^−/−-PSC tumors were followed until the study end-point was reached. Animal survival was plotted using Kaplan-Meir method. n=at least 10 in each group. D. Lack of p50 in stromal cells leads to reduced growth of another pancreatic cancer cell Panc02. Panc02 cells were either injected alone or co-injected with PSC isolated from WT or p50^−/− mice into the pancreas of WT-mice and followed for 30 days. n=at least 9 mice each group. *p<0.05. E. & F. The phenomenon that lack of NFKB1 in tumor stroma reduces tumor growth is observed in Melanoma and Lung cancer as well. E. Melanoma cell line B16-F10 (n=at least 6 mice in each group) or F. Lung cancer cell line LLC (n=at least 6 mice in each group) was injected, either alone, or with dermal or lung fibroblast respectively, from WT or p50^−/− mice subcutaneously in a WT-mice. The mice were sacrificed after 30 days and the tumor weights were compared. *p<0.05. See also Suppl. Figure 1

Figure. 2. Nfkb1 deletion in stroma reduces cellular proliferation and increases cell death and open vessel density in pancreatic tumors.

(A) Representative images and quantification of immunofluorescence evaluation of BrDu incorporation in pancreatic tumors harvested from mice injected with KPC, either alone (KPC) or co-injected with WT (KPC+WT-PSC) or p50^−/−-PSC (KPC+p50^−/−-PSC) and allowed to grow for 15 days. (B) Representative images and quantification of TUNEL staining in different groups. DAPI is used as a nuclear stain. (C) Representative images and quantification of functional vessels in the study groups, as determined by terminal perfusion with biotin-labelled tomato lectin. Quantification was performed at 20X magnification and is average of 10 random fields per mouse with data from at least 5 animals included. Data is presented mean ± SE; *P< 0.05, non-parametric Mann-Whitney test.

Figure. 3. NFKB1 depletion in tumor stroma leads to reversal of immuno-suppressive environment.

KPC pancreatic cancer cells were injected into the pancreas of C57BL/6 mice (WT), either alone (KPC) or co-injected with WT-PSC (KPC+WT) or p50^−/− PSC (KPC+p50^−/−). The mice were sacrificed after day and the infiltration of various immune cell populations into the tumors was evaluated. Representative flow cytometry plots are shown and quantification is of data from at least 5 animals in each group. (A) Lack of p50 in stroma leads to marked increase in CD8⁺ T cell infiltration and decreased (B) granulocytic MDSCs (Ly6G⁺ and CD11b⁺) as well as (C) regulatory T cells (CD4⁺ and FoxP3^+ve). (D) IHC was used to confirm the impact of p50 deletion in tumor stroma on CD8⁺ infiltration. Lack of p50 in tumor stroma also led to decreased (E) CTLA4⁺ (T cell exhaustion marker) and increased expression of markers for activated T cells including (F) Granzyme B, (G) TNF-α, (H) IFN-γ and (I) CD107a. *p<0.05

Figure. 3. NFKB1 depletion in tumor stroma leads to reversal of immuno-suppressive environment.

KPC pancreatic cancer cells were injected into the pancreas of C57BL/6 mice (WT), either alone (KPC) or co-injected with WT-PSC (KPC+WT) or p50^−/− PSC (KPC+p50^−/−). The mice were sacrificed after day and the infiltration of various immune cell populations into the tumors was evaluated. Representative flow cytometry plots are shown and quantification is of data from at least 5 animals in each group. (A) Lack of p50 in stroma leads to marked increase in CD8⁺ T cell infiltration and decreased (B) granulocytic MDSCs (Ly6G⁺ and CD11b⁺) as well as (C) regulatory T cells (CD4⁺ and FoxP3^+ve). (D) IHC was used to confirm the impact of p50 deletion in tumor stroma on CD8⁺ infiltration. Lack of p50 in tumor stroma also led to decreased (E) CTLA4⁺ (T cell exhaustion marker) and increased expression of markers for activated T cells including (F) Granzyme B, (G) TNF-α, (H) IFN-γ and (I) CD107a. *p<0.05

Figure. 4. Lack of NFκB in tumor stroma inhibits growth of tumors in CD8 dependent fashion.

(A) KPC pancreatic cancer cell line was injected into the pancreas of Rag1^−/− mice, either alone (KPC) or co-injected with WT-PSC (KPC+WT) or p50^−/− PSC (KPC+p50^−/−) and allowed to grow for 20 days. In the absence of adaptive immune system, lack of NFKB1 in tumor stroma was unable to inhibit tumor growth. (n=at least 6/group; *p<0.05) (B) KPC pancreatic cancer cell line was injected into the pancreas of C57BL/6 mice, either alone (KPC) or co-injected with WT-PSC (KPC+WT) or p50^−/− PSC (KPC+p50^−/−). After tumor initiation, mice were randomized into isotype or CD8⁺ depletion antibody injection. Antibody was administered twice weekly for 21 days. In the absence of CD8⁺ cells, lack of NFKB1 in the tumor stroma was unable to inhibit tumor growth. (n=at least 8 in each group, *p<0.05). (C) The ability of primed T cells to infiltrate towards cancer cells was evaluated in an assay similar to the Boyden chamber assay. Briefly, T cells were placed in transwells and KPC pancreatic cancer cells were placed in the lower chamber, either alone or in co-culture with WT and p50^−/− PSC. Co-culture was continued for 90 minutes, transwell membranes were harvested, stained with crystal violet and T cell migration quantified and compared between various groups. Results represent data from three independent experiments. *p<0.05 (D) The ability of primed T cells, obtained from mice bearing KPC pancreatic tumors to induce cell death in KPC cancer cells, when co-cultured with cancer cells alone or when co-cultured with cancer cells and WT or p50^−/− PSCs for 48h, was compared. Presence of p50^−/− PSCs markedly increased ability of primed CD8⁺ cells to kill cancer cells. Results represent data from three independent experiments. *p<0.05. E. CXCL12 (SDF-1) expression levels in WT and p50^−/− PSCs, when co-cultured with KPC pancreatic cancer cells in transwell system for 48h. n=3.

Figure. 5. NFKB1 dependent SDF-1 (CXCL12) Secretion from Stroma Inhibits Cytotoxic T Cell Infiltration and Promotes Tumor Growth.

A. Secretion of SDF-1(CXCL12, ng/ml), as measured by ELISA, from KPC pancreatic cancer cells when cultured alone, or when co-cultured with WT or p50^−/− PSCs. *p<0.05. B. Immunofluorescence showing the expression of SDF-1 in KPC, KPC+WT-PSC and KPC+p50^−/−-PSC tumors. C. Effect of SDF-1(CXCL12) inhibition (SDF-1/CXCL12 Neutralizing antibody) on CD8⁺ T cell migration towards KPC pancreatic cancer cells cultured alone or co-cultured with WT or p50^−/− PSC in a transwell system. WT and p50^−/− PSCs were pretreated with SDF-1/CXCL12 neutralizing antibody for 48 h, followed by addition of KPC cancer cells and T cells. D. KPC cells were injected orthotopically along with WT and p50^−/− PSC in WT background mice, on day 2 AMD3100 (SDF-1/CXCL12 inhibitor) was administered intraperitoneally daily for 21 days and tumor weights were measured after 30 days (n=at least 8 in each group, *P<0.05). E. Flow cytometry analysis showed CD8⁺ cell infiltration in tumors. (n=5/group, *P<0.05).

Figure 6:

Schematic representation summarizing the role of stromal NFκB in pancreatic cancer.