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. 2016 Apr 19;7(16):22174-85.
doi: 10.18632/oncotarget.7907.

Modulation of chemokines in the tumor microenvironment enhances oncolytic virotherapy for colorectal cancer

Lily Francis  1   2 , Zong Sheng Guo  1   2 , Zuqiang Liu  1   2 , Roshni Ravindranathan  1   2 , Julie A Urban  1   2 , Magesh Sathaiah  1   2 , Deepa Magge  1   2 , Pawel Kalinski  1   2   3   4 , David L Bartlett  1   2
Affiliations

Modulation of chemokines in the tumor microenvironment enhances oncolytic virotherapy for colorectal cancer

Lily Francis et al. Oncotarget. .

Abstract

An oncolytic poxvirus such as vvDD-CXCL11 can generate potent systemic antitumor immunity as well as targeted oncolysis, yet the antitumor effect is limited probably due to limited homing to and suppressed activity of tumor-specific adaptive immune cells in the tumor microenvironment (TME). We reasoned that a chemokine modulating (CKM) drug cocktail, consisting of IFN-α, poly I:C, and a COX-2 inhibitor, may skew the chemokine (CK) and cytokine profile into a favorable one in the TME, and this pharmaceutical modulation would enhance both the trafficking into and function of antitumor immune cells in the TME, thus increasing therapeutic efficacy of the oncolytic virus. In this study we show for the first time in vivo that the CKM modulates the CK microenvironment but it does not modulate antitumor immunity by itself in a MC38 colon cancer model. Sequential treatment with the virus and then CKM results in the upregulation of Th1-attracting CKs and reduction of Treg-attracting CKs (CCL22 and CXCL12), concurrent with enhanced trafficking of tumor-specific CD8+ T cells and NK cells into the TME, thus resulting in the most significant antitumor activity and long term survival of tumor-bearing mice. This novel combined regimen, with the oncolytic virus (vvDD-CXCL11) inducing direct oncolysis and eliciting potent antitumor immunity, and the CKM inducing a favorable chemokine profile in the TME that promotes the trafficking and function of antitumor Tc1/Th1 and NK cells, may have great utility for oncolytic immunotherapy for cancer.

Keywords: chemokine; immunotherapy; oncolytic virus; pharmaceutical modulation; tumor microenvironment.

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Conflict of interest statement

DLB is a shareholder of SillaJen Biotherapeutics, Inc. All of other authors declare no conflict of interests.

Figures

Figure 1
Figure 1. Tumor growth in CXCR3 knockout and wild type C57BL/6 mice
A. Mice were injected i.p. with 5.0E5 MC38-luc cells on day 0. Whole animal live optical imaging was performed on various days as described in Materials and methods. Shown are images on day 15 (above panel), representative picture of tumor nodules in the peritoneal cavity (lower panel). B. The difference of tumor sizes was reflected in both the tumor luminescence (p< 0.05) and tumor burden (p= 0.07). C. The survival of tumor-bearing CXCR3 KO and wild type (WT) mice are plotted demonstrating a significant difference in length of survival (p < 0.001).
Figure 2
Figure 2. The new virus vvCXCL11 was functional as an oncolytic virus and expressed CXCL11 from infected cancer cells in vitro and in vivo
A. Schematic representation of the new virus vvCXCL11 and parental virus vvDD (full name vvDD-CD). B. Viral replication in MC38 cancer cells. C. Expression and secretion of CXCL11 from infected MC38 cancer cells. CXCL11 in the conditioned medium was quantified by an ELISA assay. D. The levels of CXCL11 in tumor tissues were quantified by making the tumor tissue lysate and measured via ELISA assay. P value: ***, p < 0.001.
Figure 3
Figure 3. CXCL11 expressed from the virus enhances infiltration of CD8+ T Cells into the tumor tissues, but yields no significant survival benefit
A. IHC of CD8+ cells in the tumor tissues. The control virus is vvDD and CXCL11 virus is vvDD-CXCL11. B. Real-time RT-PCR for mRNAs encoding CD8 (marker for CD8+ T cells and CD8+ DC) and NKG2D (activation marker on NK and T cells). Two days after virus treatment, tumor tissues were harvested and the total RNA was purified from the tissues. C. Kaplan-Meier plot for survival of tumor-bearing mice treated with PBS, vvDD, or vvDD-CXCL11. The p values are indicated: *p <0.05; **p < 0.01; ns = not significant.
Figure 4
Figure 4. Two doses of CKM enhanced CD3 CD8+ markers and CXCL11, but not granzyme B levels in the tumor tissue, and did not enhance the survival of MC38-luc tumor-bearing C57BL/6 mice
A. Shown are qRT-PCR results on the levels of mRNAs for CD3, CD8, granzyme B (GzmB), and CXCL11 from tumor tissue after treatment with PBS, 1 or 2 doses of CKM. Tumor tissues were harvested at 48 h post final treatment and purified total RNA was used for RT-qPCR. p = ns, not significant; * p< 0.05; **p< 0.01. B. Kaplan-Meier analysis of survival of mice bearing MC38-luc tumor treated with PBS or CKM for a total of 6 doses. p = ns.
Figure 5
Figure 5. CKM modulates the TME into one with an immunostimulatory CK profile, enhances the trafficking of CD4 and CD8 T cells and potentiates killer activity
A. Real-time PCR analyses on levels of mRNAs for cytokine and CK markers in the TME. Tumor tissues were harvested at 48 h post final treatment and purified total RNA was used for RT-qPCR. Included are pro-inflammatory CKs (CCL5, CXCL9 and CXCL11), and cytokines IFN-γ and IL-10. B. Immune cell markers CD4, CD8, killer cell activation marker granzyme B (GzmB) and NKG2D, as well as the mRNA level for COX-2. C. Treg cell-attracting CKs (CCL22 and CXCL12) and Treg cell marker FoxP3. RT-qPCR was performed as described in Materials and Methods. The mRNA levels were then expressed as relative quantity in relative to the house-keeping gene HPRT (Fold over HPRT). Data are representatives from two independent experiments. The virus used is vvDD-CXCL11 and labeled just as “virus” for simplicity. Standard symbols are used to indicate p values: *p < 0.05; **p < 0.01.
Figure 6
Figure 6. The virus vvCXCL11, but not CKM, has major effects on the systemic immunity, and dual treatment synergistically prolongs the survival of MC38-luc tumor-bearing mice
A. Scheme of the treatments of mice and immunological assays. B. IFN-γ ELISPOT assay on the splenocytes collected on day 5 after final treatment. p <0.001 (***) when comparing CKM to the virus or virus+CKM; p = ns when comparing virus to virus+ CKM. C. Total amounts of secreted IFN-γ from cocultures of splenocytes with irradiated MC38-luc cells incubated for 48 hr. D. Kaplan-Meier survival analysis of MC38-luc carcinomatosis-bearing C57BL/6 mice treated with PBS, CKM, vvDD-CXCL11, or the combination (n=10 for each group). The median survival are, 28 days for PBS or CKM, 48 days for vvDD-CXCL11, at least 115 days for the combination (p<0.001 between singular versus the combination treatments).
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