Clinical significance and therapeutic implication of CD200 in pancreatic cancer

Background CD200, a negative T cell regulator as well as a cancer stem cell marker, is a significant prognostic factor and potential therapeutic target in specific cancers. However, the clinical significance of CD200 is unknown in pancreatic ductal adenocarcinoma (PDAC). CD200 was evaluated in 220 resected PDAC patients. Surgery was performed with or without neoadjuvant chemotherapy (NACRT), and adjuvant therapy was administered with systemic therapy or systemic therapy added with hepatic arterial infusion (HAI) therapy. We investigated the clinicopathological outcomes associated with CD200, in relation to the administered multimodal treatment. We further evaluated the impact of the immunological and cancer stem cell properties associated with CD200.

manufacturer's instructions, then counterstained with hematoxylin. CD200 and CD44s expression were evaluated by inspection of 10 random fields in ×200 magnification within the proximity of cancerous areas. A small, oval or spindle-shaped cell with a large nucleus, which showed cytoplasm staining of CD200 with an intensity similar to islet cells, was observed in the peripheral of some cancer cell lesions. The intensity of this cell was uniformly strong. There were differences in the ratio of positive fields, but regardless of distribution, patients who displayed these positive cells were considered as CD200 positive PDAC cases. For evaluation of CD44, the variant isoform CD44s was selected. CD44+ PDAC was defined with similar criteria as CD200+ PDAC. A cell with a shape similar to CD200 positive PDAC, which displayed membranous staining was observed in some cases. Were considered these cases as CD44 positive PDAC. For tumor-infiltrating lymphocyte (TIL) evaluation, 5 random fields in ×200 magnification with clustered lymphocytes in the proximity of cancerous lesions were investigated, and the mean lymphocyte count was documented.

Cell lines and culture
The human pancreatic cancer cell lines PANC-1 and Capan-2 were obtained from Riken BioResource Research Center (Tsukuba, Japan) and DS Pharma Biomedical (Suita, Japan) respectively, and cultured in RPMI-1640 supplemented with 10% heat-inactivated fetal bovine serum. Irradiation 4.0×10 5 cells were seeded in 10 cm culture dishes. After overnight incubation, the monolayer cultures were irradiated with 10 Gy in a single fraction using a 150-KVp X-ray generator (MBR-1520R, Hitachi, Kashiwa, Japan) at a dose rate of 1.0 Gy/min. Following irradiation, cells were incubated for 7 days in the same culture medium.

Flow cytometry analysis
The expression of CD200, CD44, CD24 and EpCAM of irradiated and control cells were analyzed by FACSCalibur and CellQuest Pro software (BD Biosciences, Franking Lakes, New Jersey, USA). The staining of these markers was performed following the manufacturer's protocol. Briefly, a single-cell suspension was prepared by trypsinization, and then cells were incubated with monoclonal antibodies (mAbs) for 25 min at room temperature after fixation with 4% paraformaldehyde for 5 min. The following mAbs were used; fluorescein isothiocyanate (FITC)-labeled anti-CD24, allophycocyanin (APC)-labeled anti-CD44, APClabeled anti-epithelial cell adhesion molecule (EpCAM) (Biolegend, San Diego, California, USA), phycoerythrin (PE)-labeled anti-CD200 (Beckman Coulter, Brea, California, USA), and isotype controls for each mAbs. The viability of cells was tested by 7-amino-acitomycin D staining and was approximately 98% in each experiment. The isotype control staining defined the quadrant area. (hazard ratio (HR) = 0.514, 95% confidence interval (CI) 0.297-0.892, P = 0.018) and distant metastasis were found to be independent prognostic factors (Additional file 1: Table S1).

Prognostic impact of CD200 in PDAC in association with pre-operative treatment
First, we evaluated CD200 expression in whole tissue specimens by IHC. The staining of CD200 was consistently intense in a small, round to spindle cell found in the peripheral of cancer lesions (Fig. 1a). Patients positive of this cell were defined as CD200+ PDAC. The CD200 positivity rate was significantly higher in NACRT (n = 67, 66.4%), compared with US patients (n =37, 32.2%) (P < 0.001; Fig. 1b). Furthermore, CD200 positive cells detected in half or more of the inspected fields were significantly greater in NACRT (n = 31, 46.3%) than US (n = 3, 8.1%) (P < 0.0001). In US patients, there was no difference in postoperative survival between CD200+ and CD200-cases (21.9 M vs. 30.6 M, P = 0.31; Fig. 1c). On the contrary, in NACRT patients, CD200+ cases had a significantly poorer prognosis than CD200-(33.4 M vs. 57.8 M, P = 0.03; Fig. 1d). Subsequently, we investigated the clinicopathological factors associated with poor prognosis in NACRT patients. Among the baseline patient demographics, RECIST response was the only significant difference associated with CD200 expression ( Table 2). A univariate and multivariate Cox regression analysis of NACRT patients showed CD200 expression to be an independent poor prognostic factor (HR = 2.51, 95% CI 1.35-4.66, P = 0.004), as well as resection status, post-NACRT CA19-9, and distant metastasis (Table 3). However, in univariate and multivariate analysis of all patients, CD200 was not a prognostic factor.
To evaluate why CD200 lacked prognostic value in US patients, we assessed the differences in patient background. Since HAI was an independent prognostic factor in all patients, the significant difference in HAI administration rate between US and NACRT patients needed consideration. Therefore, we evaluated the prognostic impact of CD200 in a subgroup of patients only which received HAI therapy. In this subgroup, there was no

Potential immunological impact of CD200
To investigate the mechanisms of poor prognosis in CD200+ patients after NACRT, we first looked into the association between CD200 and immunological factors.

Correlation of CD200 with pancreatic cancer CSC marker
Since CD200 has been reported to co-express in CSCs [19], we next considered whether CSC properties were associated with the poor prognosis in CD200+ NACRT patients. . Therefore, as an initial investigation, we evaluated the correlation of CD200 with CD44s expression by IHC in whole tissue samples of NACRT patients.

Influence of irradiation to CD200 and CSC marker expression in human pancreatic cancer cells
Next, since the CD200 expression rate increased after NACRT in the clinical setting, we investigated the role of irradiation on CD200 expression in an in vitro model. Also, we sought to verify our hypothesis of CD200 and CSC marker correlation by cell-surface marker analysis of CD200, CD44, CD24, and ESA with flow cytometry in human PDAC cells.
After a single-fraction irradiation of 10Gy, the CD200 positivity rate increased from 0.5% in control to 13.5% in irradiated PANC-1 cells, and from 1.7% in control to 17.5% in irradiated Capan-2 cells (Fig. 3c). Regarding CSC markers, post-irradiation CD200+ cells showed a stronger association with CSC markers after irradiation, compared with postirradiation CD200-or control cells (Fig. 3d). The CD44+/CD24+ double-positive rate was higher after irradiation in CD200+ cells in both PANC-1 and Capan-2 cells. Although ESA expression was high (>96 %) regardless of irradiation in both PANC-1 and Capan-2 cells, CD200+ had a higher ESA+ rate compared to CD200-, in both control and irradiated cells (data of ESA expression from control cells not shown).

Impact of CD200 on the post-operative recurrence patterns
Finally, we investigated the association between CD200 and post-operate recurrence patterns. Since we have reported the preventive effect of HAI against hepatic recurrence [27], and also, since hepatic recurrence generally has a more unsatisfactory clinical outcome compared with other recurrence types, we especially paid attention to the hepatic recurrence patterns. The total hepatic recurrence rate did not differ between US (19.1%, n = 22) and NACRT (22.9%, n = 24) (P = 0.51). Among the patients who received post-operative treatment, the hepatic recurrence rate was lower after HAI (6.1 %, n = 7 ) than systemic therapy (11.3 %, n = 13) in US (P = 0.05), but was similar (15.2 %, n = 16 vs. 5.7 %, n = 6) in NACRT (P = 0.37). The rate of other failure patterns did not differ between HAI and systemic therapy in either NACRT or US patients (Additional file 2: Table   S2).
Likewise, the association between CD200 and hepatic recurrence according to adjuvant therapy regimen was investigated in US patients. As a result, the proportion of hepatic recurrence was lower after HAI compared to systemic therapy, regardless of CD200 expression (Fig. 4c). Hepatic recurrence occurred in 1 out of 9 (11.1%) of HAI/CD200+, 6 out of 30 (20.0%) of HAI/CD200-, 5 out of 16 (31.3%) of systemic/CD200+, and 8 out of 21 (38.1%) of systemic/CD200-patients with recurrence.

Discussion
Immunotherapy, namely ICB therapy, in combination with conventional modalities, such as chemotherapy, radiotherapy (RT) or surgery, is a potential novel treatment strategy. RT is We have been treating borderline resectable PDAC, as well as resectable PDAC with multimodal treatment by NACRT and adjuvant HAI therapy [26]. In this study, to identify insights into novel combination immunotherapy strategies, we set to retrospectively investigate the clinical impact of a T cell negative-regulator CD200 under the various circumstances of multimodal therapy in our series. As a result, CD200 seemed activated by NACRT and displayed association with suppression of local tumor immunity and therapy resistance to HAI.
From the results of IHC evaluation, we have shown for the first time that CD200 had a significant prognostic impact on PDAC. CD200 was associated with poor prognosis in post-NACRT patients, independent of widely known clinical factors including NCCN resectability status, pre-operative CA19-9 levels, and distant metastasis. We suspected that the suppressive immunological effect of CD200 was a potential mechanism of poor prognosis. CD200 co-expression with CSC markers has been reported in prostate, breast, brain, and colon cancer [46]. In this study, we have shown for the first time that CD200 expression correlated with pancreatic cancer CSCs. As a result of irradiation, CD44+/CD24+/ESA+ expressing cell were enriched, suggesting that cells presenting CSC properties were activated or derived from irradiation stimulus. Since the correlation with CSC markers in both CD200+ and CD200-cells changed after irradiation, we assumed that the cells presenting PDAC CSC markers were not the result of the expansion of cells which were resistant to irradiation, but rather, that they were derived from irradiation stimulus.
Data from preclinical studies show that CSC properties such as tumor initiation, therapy resistance, and metastasis promotion are not unique to CSCs, but are also seen in other cells such as those that underwent epithelial-mesenchymal transition (EMT). Mani et al. From our clinical data, CD200+ NACRT patients appeared to be refractory to HAI therapy, suggesting that therapy resistance resulting from CSC properties of CD200 may have contributed to the poor prognosis. Furthermore, the survival of CD200-post-NACRT patients was significantly better, while the survival of CD200+ post-NACRT patients was similar to the SF patient cohort. Therefore, in CD200+ NACRT patients, although NACRT was associated with higher local tumor control, the resistance to post-operative therapy may have caused a trade-off in prognostic benefit, to result in survival similar to the US approach. In the present study, CD200 was not evaluated before NACRT administration, so the direct change of CD200 expression could not be observed in the individual patients. In future studies, elucidation of the prediction of CD200 expression after NACRT could help to predict the optimal indication for NACRT in PDAC. Furthermore, since CD200-NACRT patients showed a remarkable prognosis, it was suggested that therapy resistance associated with CD200 expression might have a vital clinical significance in PDAC with chemoradiation, and strategies to eradicate CD200 may be a key step to improve the efficacy of multidisciplinary therapy in pancreatic cancer.

Conclusions
In summary, we have shown the novel roles of CD200 in immune evasion and therapy resistance in PDAC. CD200 may play a critical role in the modulation of tumor immunity and therapy resistance under irradiation stimulus; therefore, it was suggested that CD200 might be a potential target in combination with RT in PDAC. Although further mechanistic investigations are required, this study provides valuable insights into establishing novel methods to improve the efficacy of multidisciplinary treatment against human pancreatic cancer.