Purpose: Cancer-associated fibroblasts (CAFs) in the tumor microenvironment (TME) play a central role in tumor progression. We investigated whether CAFs can regulate tumor-infiltrating lymphocytes (TILs) and their role in tumor immunosuppression. Experimental Design: 140 cases of esophageal cancer were analyzed for CAFs and CD8⁺or forkhead box protein 3 (FoxP3⁺) TILs by immunohistochemistry. We analyzed cytokines using murine or human fibroblasts and cancer cells. Murine-derived fibroblasts and cancer cells were also inoculated into BALB/c or BALB/c-nu/numice, and the tumors treated with recombinant interleukin 6 (IL-6) or anti-IL-6 antibody. Results: CD8⁺TILs and CAFs were negatively correlated in intra-tumoral tissues (P< 0.001), while FoxP3⁺TILs were positively correlated (P< 0.001) in esophageal cancers. Co-cultured Colon26 cancer cells and fibroblasts resulted in accelerated tumor growth in BALB/c mice, along with decreased CD8⁺and increased FoxP3⁺TILs, compared with cancer cells alone. In vitro, IL-6 was highly secreted in both murine and human cancer cell/fibroblast co-cultures. IL-6 significantly increased Colon26 tumor growth in immune-competent BALB/c (P< 0.001) with fewer CD8⁺TILs than untreated tumors (P< 0.001), whereas no difference in BALB/c-nu/numice. In contrast, FoxP3⁺TILs increased in IL-6-treated tumors (P< 0.001). IL-6 antibody blockade of tumors co-cultured with fibroblasts resulted not only in regression of tumor growth but also in the accumulation of CD8⁺TILs in intra-tumoral tissues. Conclusions:CAFs regulate immunosuppressive TIL populations in the TME via IL-6. IL-6 blockade, or targeting CAFs, may improve pre-existing tumor immunity and enhance the efficacy of conventional immunotherapies.

Evaluation of the blood supply to gastric conduits is critically important to avoid complications after esophagectomy. We began visual evaluation of blood flow using indocyanine green (ICG) fluorescent imaging in July 2015, to reduce reconstructive complications. In this study, we aimed to statistically verify the efficacy of blood flow evaluation using our simplified ICG method. A total of 285 consecutive patients who underwent esophagectomy and gastric conduit reconstruction were reviewed and divided into 2 groups: before and after introduction of ICG evaluation. The entire cohort and 68 patient pairs after propensity score matching (PS-M) were evaluated for clinical outcomes and the effect of visualized evaluation on reducing the risk of complication. The leakage rate in the ICG group was significantly lower than in the non-ICG group for each severity grade, both in the entire cohort (285 subjects) and after PS-M; the rates of other major complications, including recurrent laryngeal nerve palsy and pneumonia, were not different. The duration of postoperative ICU stay was approximately 1 day shorter in the ICG group than in the non-ICG group in the entire cohort, and approximately 2 days shorter after PS-M. Visualized evaluation of blood flow with ICG methods significantly reduced the rate of anastomotic complications of all Clavien-Dindo (CD) grades. Odds ratios for ICG evaluation decreased with CD grade (0.3419 for CD ≥ 1; 0.241 for CD ≥ 2; and 0.2153 for CD ≥ 3). Objective evaluation of blood supply to the reconstructed conduit using ICG fluorescent imaging reduces the risk and degree of anastomotic complication.

Cancer-associated fibroblasts (CAFs) are strongly implicated in tumor progression, including in the processes of tumorigenesis, invasion, and metastasis. The targeting of CAFs using various therapeutic approaches is a novel treatment strategy; however, the efficacy of such therapies remains limited. Recently, near-infrared photoimmunotherapy (NIR-PIT), which is a novel targeted therapy employing a cell-specific mAb conjugated to a photosensitizer, has been introduced as a new type of phototherapy. In this study, we have developed a novel NIR-PIT technique to target CAFs, by focusing on fibroblast activation protein (FAP), and we evaluate the treatment efficacy in vitro and in vivo. Esophageal carcinoma cells exhibited enhanced activation of fibroblasts, with FAP over-expressed in the cytoplasm and on the cell surface. FAP-IR700-mediated PIT showed induced rapid cell death specifically for those cells in vitro and in vivo, without adverse effects. This novel therapy for CAFs, designed as local control phototherapy, was safe and showed a promising inhibitory effect on FAP⁺ CAFs. PIT targeting CAFs via the specific marker FAP may be a therapeutic option for CAFs in the tumor microenvironment in the future.

Cancer-associated fibroblasts (CAFs) have an important role in the tumor microenvironment. CAFs have the multifunctionality which strongly support cancer progression and the acquisition of therapeutic resistance by cancer cells. Near-infrared photoimmunotherapy (NIR-PIT) is a novel cancer treatment that uses a highly selective monoclonal antibody (mAb)-photosensitizer conjugate. We developed fibroblast activation protein (FAP)-targeted NIR-PIT, in which IR700 was conjugated to a FAP-specific antibody to target CAFs (CAFs-targeted NIR-PIT: CAFs-PIT). Thus, we hypothesized that the control of CAFs could overcome the resistance to conventional chemotherapy in esophageal cancer (EC). In this study, we evaluated whether EC cell acquisition of stronger malignant characteristics and refractoriness to chemoradiotherapy are mediated by CAFs. Next, we assessed whether the resistance could be rescued by eliminating CAF stimulation by CAFs-PIT in vitro and in vivo. Cancer cells acquired chemoradiotherapy resistance via CAF stimulation in vitro and 5-fluorouracil (FU) resistance in CAF-coinoculated tumor models in vivo. CAF stimulation promoted the migration/invasion of cancer cells and a stem-like phenotype in vitro, which were rescued by elimination of CAF stimulation. CAFs-PIT had a highly selective effect on CAFs in vitro. Finally, CAF elimination by CAFs-PIT in vivo demonstrated that the combination of 5-FU and NIR-PIT succeeded in producing 70.9% tumor reduction, while 5-FU alone achieved only 13.3% reduction, suggesting the recovery of 5-FU sensitivity in CAF-rich tumors. In conclusion, CAFs-PIT could overcome therapeutic resistance via CAF elimination. The combined use of novel targeted CAFs-PIT with conventional anticancer treatments can be expected to provide a more effective and sensible treatment strategy.

The programmed cell death 1 protein (PD-1)/programmed cell death ligand 1 (PD-L1) axis plays a crucial role in tumor immunosuppression, while the cancer-associated fibroblasts (CAFs) have various tumor-promoting functions. To determine the advantage of immunotherapy, the relationship between the cancer cells and the CAFs was evaluated in terms of the PD-1/PD-L1 axis. Overall, 140 cases of esophageal cancer underwent an immunohistochemical analysis of the PD-L1 expression and its association with the expression of the α smooth muscle actin, fibroblast activation protein, CD8, and forkhead box P3 (FoxP3) positive cells. The relationship between the cancer cells and the CAFs was evaluated in vitro, and the effect of the anti-PD-L1 antibody was evaluated using a syngeneic mouse model. A survival analysis showed that the PD-L1⁺ CAF group had worse survival than the PD-L1^- group. In vitro and in vivo, direct interaction between the cancer cells and the CAFs showed a mutually upregulated PD-L1 expression. In vivo, the anti-PD-L1 antibody increased the number of dead CAFs and cancer cells, resulting in increased CD8⁺ T cells and decreased FoxP3⁺ regulatory T cells. We demonstrated that the PD-L1-expressing CAFs lead to poor outcomes in patients with esophageal cancer. The cancer cells and the CAFs mutually enhanced the PD-L1 expression and induced tumor immunosuppression. Therefore, the PD-L1-expressing CAFs may be good targets for cancer therapy, inhibiting tumor progression and improving host tumor immunity.