LIGHT Elevation Enhances Immune Eradication of Colon Cancer Metastases

Abstract

The majority of patients with colon cancer will develop advanced disease, with the liver being the most common site of metastatic disease. Patients with increased numbers of tumor-infiltrating lymphocytes in primary colon tumors and liver metastases have improved outcomes. However, the molecular factors that could empower antitumor immune responses in this setting remain to be elucidated. We reported that the immunostimulatory cytokine LIGHT (TNFSF14) in the microenvironment of colon cancer metastases associates with improved patient survival, and here we demonstrate in an immunocompetent murine model that colon tumors expressing LIGHT stimulate lymphocyte proliferation and tumor cell-specific antitumor immune responses. In this model, increasing LIGHT expression in the microenvironment of either primary tumors or liver metastases triggered regression of established tumors and slowed the growth of liver metastases, driven by cytotoxic T-lymphocyte-mediated antitumor immunity. These responses corresponded with significant increases in tumor-infiltrating lymphocytes and increased expression of lymphocyte-homing signals in the metastatic tumors. Furthermore, we demonstrated evidence of durable tumor-specific antitumor immunity. In conclusion, increasing LIGHT expression increased T-cell proliferation, activation, and infiltration, resulting in enhanced tumor-specific immune-mediated tumor regressions in primary tumors and colorectal liver metastases. Mechanisms to increase LIGHT in the colon cancer microenvironment warrant further investigation and hold promise as an immunotherapeutic strategy. Cancer Res; 77(8); 1880-91. ©2017 AACR.

Figure 1. LIGHT expressing colon cancer cell lines maintain wild-type characteristics

(A) To construct constitutive and doxycycline inducible(i) expressing LIGHT lentiviral vectors, murine LIGHT cDNA was cloned into MluI/PstI sites of the pHIV-Puro (pHIV-LIGHT) and pLVX-TRE3G (pLVX-LIGHT) parental plasmids. (B) CT26 cell clones constitutively expressing LIGHT (CT26LIGHT) or inducible expressing LIGHT (CT26LIGHTⁱ) or its empty control (CT26controlⁱ) were established after drug selection and limited dilution, and LIGHT expression was analyzed using FACS after DNA sequencing. (C) LIGHT protein expression was confirmed in CT26LIGHT cells and CT26LIGHTⁱ cells in the presence of doxycycline. The LIGHT expressing murine fibrosarcoma cell line Ag104LIGHT served as a positive control. (D) wtCT26, CT26control (empty vector), CT26controlⁱ, CT26LIGHTand CT26LIGHTⁱ cells were evaluated for proliferation, apoptosis, and migration. Representative experiments are shown of the constitutive expressing cell line (see also supplemental figure 1). Cells were seeded in triplicate and MTS/PMS solution was added on days 1 2, 3, 4, and 5 with no difference in cell proliferation or metabolic activity.

Figure 2. Recombinant LIGHT and LIGHT expressing colon cancer cells enhance T-cell and dendritic cell activation in vitro

(A,B) CSFE-labeled T-cells were added to round-bottom 96-well plates coated with anti-CD3 and rLIGHT. CD4 and CD8 T-cell proliferation increased with increasing doses of rLIGHT. Shown are representative results of three independent experiments. (C) Bone marrow derived dendritic cells (DC) were exposed to 100–1000ng/mL of soluble LIGHT or LPS as a positive control. Significantly increased DC cell activation was observed with increasing doses of soluble LIGHT. (D) Purified T-cells were cocultured with CT26Control or CT26LIGHT cells revealing increased cell proliferation in the presence of LIGHT that was inhibited by the addition of LTbR-Ig mediated blockade of LIGHT's cognate receptor. (E,F) Cell culture supernatants were collected after 48 hours and IFNγ was detected by ELISA. Increased T-cell IFNγ production was observed in the presence of CT26LIGHT compared to CT26control. (F) Blocking LIGHT with LTbR-Ig decreased T-cell IFNγ production compared to control Ig. (* p<0.05, **p<0.01).

Figure 3. LIGHT expressing tumors generated increased T-cell proliferation, a tumor-specific anti-tumor immune response, and significant T-cell mediated tumor regressions

(A) Primary flank colorectal cancer cell tumors were established in BALB/c mice. Three weeks after tumor inoculation splenocytes were harvested (n=15). Mice with induced LIGHT expression (+ doxycycline diet) demonstrated increased numbers of CD3, CD4, CD8, and CD11c+ cells compared to CT26LIGHTⁱ tumors fed normal diet and CT26controlⁱ tumors fed doxycycline diet. (B) Splenocytes from animals with established colon cancer flank tumors were then used as effector cells, and their cytolytic capacities were assessed against ⁵¹Cr-labeled 4T1 cancer cells or wtCT26 tumor cells. Only lymphocytes from animals with LIGHT expressing CT26 tumors demonstrated tumor-specific cytotoxicity against wtCT26 and not 4T1 cells. (C) CT26LIGHTⁱ colon cell tumors were established in animals fed normal diet (n=18) and once palpable and measurable, randomized on day 6 (arrow) to doxycycline diet (LIGHT induction) or normal diet. Significant tumor regression began only in animals with LIGHT induction and continued with partial and complete tumor responses as measured by total tumor volume, WHO criteria, and RECIST clinical tumor response criteria (pictured). (D) Lymphocyte depletion was performed of specific immunocyte subsets. CT26LIGHTⁱ colon cell tumors were established in animals and on day 6 (arrow) received doxycycline diet (LIGHT induction) as before, confirming significant tumor regressions in animals with LIGHT induction. The anti-tumor immune response was abrogated in the absence of CD3 and CD8 T-lymphocytes.

Figure 4. LIGHT expressing tumors induced increased tumor infiltrating lymphocyte infiltration and generated persistent anti-tumor immunity

(A) Tumors were harvested 21 days after tumor inoculation (n=15). Individual 20× fields on Vectra multispectral imaging system were stitched into whole tumor images (B), the total number of CD3-positive cells and colon cancer cells in the tumor were quantified, and CD3 cells per 100 tumor cells was determined to be significantly increased in LIGHT expressing tumors (C). (D) Mice with established CT26LIGHTⁱ tumors (n=8 per group) were injected subcutaneously with 4T1 syngeneic breast cancer cells in the contralateral superior flank and with CT26LIGHTⁱ in the contralateral inferior flank. 4T1 tumors grew rapidly and to similar size in mice from both groups after the tumor challenge. On the other hand, CT26LIGHTⁱ challenge tumors from mice previously exposed to LIGHT expressing tumors grew minimally and then disappeared, while the challenge tumors in mice fed regular diet grew significantly larger. Tumor volumes of 4T1 are on the left y-axis, and volumes of CT26 are on the right y-axis. *p<0.05 and **p<0.01.

Figure 5. LIGHT inhibits colorectal liver metastases (CRLM) growth and increases metastatic tumor infiltrating lymphocytes

(A) CRLM were established with CT26LIGHT and CT26LIGHTⁱ colon cancer cells and tumor burden was followed with MRI imaging. (B) CT26LIGHTⁱ CRLM (n=7 per group) were resected on day25. Increased LIGHT expression was confirmed in CRLM tumors from animals fed doxycycline. (C) The percent of affected liver was quantified with imaging software and was significantly decreased in tumors expressing LIGHT. (D) Total tumor burden as measured with liver weight was similarly significantly decreased. (E) Peripheral blood mononuclear cells from animals harboring LIGHT expressing CRLM demonstrated increased populations of CD3, CD4, and CD8 T-cells, NK cells, and DC cells. (F) LIGHT expressing CRLM similarly recruited increased numbers of total lymphocytes, CD3 cells, NK cells, and dendritic cells into the tumor.