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. 2012 Aug 1;72(15):3735-43.
doi: 10.1158/0008-5472.CAN-11-4136. Epub 2012 May 16.

NF-κB hyperactivation in tumor tissues allows tumor-selective reprogramming of the chemokine microenvironment to enhance the recruitment of cytolytic T effector cells

Ravikumar Muthuswamy  1 , Erik Berk, Beth Fallert Junecko, Herbert J Zeh, Amer H Zureikat, Daniel Normolle, The Minh Luong, Todd A Reinhart, David L Bartlett, Pawel Kalinski
Affiliations

NF-κB hyperactivation in tumor tissues allows tumor-selective reprogramming of the chemokine microenvironment to enhance the recruitment of cytolytic T effector cells

Ravikumar Muthuswamy et al. Cancer Res. .

Abstract

Tumor infiltration with effector CD8(+) T cells (T(eff)) predicts longer recurrence-free survival in many types of human cancer, illustrating the broad significance of T(eff) for effective immunosurveillance. Colorectal tumors with reduced accumulation of T(eff) express low levels of T(eff)-attracting chemokines such as CXCL10/IP10 and CCL5/RANTES. In this study, we investigated the feasibility of enhancing tumor production of T(eff)-attracting chemokines as a cancer therapeutic strategy using a tissue explant culture system to analyze chemokine induction in intact tumor tissues. In different tumor explants, we observed highly heterogeneous responses to IFNα or poly-I:C (a TLR3 ligand) when they were applied individually. In contrast, a combination of IFNα and poly-I:C uniformly enhanced the production of CXCL10 and CCL5 in all tumor lesions. Moreover, these effects could be optimized by the further addition of COX inhibitors. Applying this triple combination also uniformly suppressed the production of CCL22/MDC, a chemokine associated with infiltration of T regulatory cells (T(reg)). The T(eff)-enhancing effects of this treatment occurred selectively in tumor tissues, as compared with tissues derived from tumor margins. These effects relied on the increased propensity of tumor-associated cells (mostly fibroblasts and infiltrating inflammatory cells) to hyperactivate NF-κB and produce T(eff)-attracting chemokines in response to treatment, resulting in an enhanced ability of the treated tumors to attract T(eff) cells and reduced ability to attract T(reg) cells. Together, our findings suggest the feasibility of exploiting NF-κB hyperactivation in the tumor microenvironment to selectively enhance T(eff) entry into colon tumors.

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Conflict of interest statement

Disclosure of Potential Conflicts of Interest: The methods of tumor-selective regulation of chemokine environment are the subject to a pending patent application by the University of Pittsburgh. None of the authors receives any form of remuneration related to these findings.

Figures

Figure 1
Figure 1. Presence of Teff- and Treg markers in tumors correlates with intra-tumoral expression of, respectively, Teff- or Treg-attracting chemokines
Tumor biopsies from colon cancer patients were lysed, RNA extracted and Taqman analysis of various markers was performed. (A) Correlation between Teff markers (CD8 and Granzyme B; GZMB) and Teff-attracting chemokines (CCL5 and CXCL10) in tumor lesions. (B) Correlation between Treg markers (FOXP3 and GITR) and the chemokine CCL22 in tumor lesions.
Figure 2
Figure 2. Heterogenous response pattern of different tumor tissues to individual chemokine modulators and their uniform response to the combination of IFNα, poly-I:C and indomethacin
(A) Fresh tumor samples from 11 patients with metastatic colorectal cancer were untreated or treated with IFNα and poly-I:C either individually or in combination for 48 hours. The release of CCL5 and CXCL10 into culture media was analyzed by ELISA. Numbers indicate the prevalence of tumors with each chemokine pattern (respective patterns A, B or C). (B) ELISA analysis of CCL5, CCL22 and CXCL10 in tumors untreated or treated with IFNα+ pI:C, with or without indomethacin. *denotes P<0.05 (the presence or absence of indomethacin).
Figure 3
Figure 3. Combination of IFNα, poly-I:C and indomethacin, consistently up regulates Teff-attracting chemokines and suppresses Treg-attracting chemokines in tumor tissues
(A) In-situ hybridization for respective chemokine mRNA (black grains) in tumor biopsies which were either left untreated or treated with the combination of indomethacin, IFNα and poly-I:C (IAP). (B) ELISA analysis of the chemokine contents in the supernatants of 48 hour-cultured tumor tissues (untreated or treated) from 10 different patients.
Figure 4
Figure 4. NF-κB-dependent selective enhancement of CXCL10 production in tumor tissues following exposure to the combination of IFNα, poly-I:C and indomethacin
(A) ELISA for CXCL10 expression in matched normal liver and liver metastatic tissues from 10 different patients either untreated (left panel) or treated (right panel). (B) Average number of cells counted per field (confocal microscopy; in a total of 10 fields) showing nuclear translocation of NF-κB in normal liver or liver metastatic tissues either untreated or treated (right panel). Representative images of each condition are shown in the left panel. (C) ELISA analysis of CXCL10 production by the matched normal liver and liver-metastatic colorectal cancer tissues, either untreated or treated (IFNα, poly-I:C and indomethacin), in the absence or presence of 20μM CAY10470 (NF-κB inhibitor). D) Colorectal cancer tissues from three colorectal cancer patients were treated with indomethacin + IFNα + Poly-I:C for 30 minutes (p65 translocation) and 24 hours (chemokine production) and analyzed by confocal microscopy for the translocation of p65 and production of CCL5 and CXCL10 by infiltrating inflammatory cells (CD45+), tumor-associated fibroblasts (TE-7-binding cells) and cancer cells (CD326). Representative data from one of three experiments. Left panel: representative sections of the activated tumor tissue. Right panel: Numbers per vision field of the individual cells types showing p65 translocation and chemokine production. Data from 10 vision fields is expressed as average +/− SEM.
Figure 5
Figure 5. IFNα, poly-I:C and indomethacin-treated tumors show enhanced ability to attract Teff, but strongly-reduced ability to attract Tregs
(A) Ex vivo generated Teff (left) or isolated CD8+ tumor-infiltrating lymphocytes (right) (see Materials and Methods) were allowed to migrate towards supernatants from either untreated or treated tumors from 3 different patients in transwell chemotaxis assays. (B) Negatively-isolated total CD4+ T cells were allowed to migrate towards the treated- or untreated tumor supernatants. Migrating cells were lysed and analyzed for FOXP3 expression by Taqman. U.D.: undetectable.
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