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. 2018 Jan;39(1):151-159.
doi: 10.3892/or.2017.6097. Epub 2017 Nov 14.

Immunogenic tumor cell death induced by chemotherapy in patients with breast cancer and esophageal squamous cell carcinoma

Keita Aoto  1 , Kousaku Mimura  2 , Hirokazu Okayama  1 , Motonobu Saito  1 , Shun Chida  1 , Masaru Noda  1 , Takahiro Nakajima  1 , Katsuharu Saito  1 , Noriko Abe  1 , Shinji Ohki  1 , Tohru Ohtake  1 , Seiichi Takenoshita  1 , Koji Kono  1
Affiliations

Immunogenic tumor cell death induced by chemotherapy in patients with breast cancer and esophageal squamous cell carcinoma

Keita Aoto et al. Oncol Rep. 2018 Jan.

Abstract

It has been reported that chemo-radiotherapy can induce immunogenic tumor cell death (ICD), which triggers T-cell immunity mainly mediated by high-mobility group box 1 protein (HMGB1) and calreticulin. However, there is still limited information to support this theory relating to chemotherapy alone. In the present study, the expression of HMGB1 and calreticulin was evaluated by immunohistochemistry in pre-treatment biopsy specimens and surgically resected specimens, which were obtained from patients with breast cancer (n=52) and esophageal squamous cell carcinoma (ESCC) (n=8) who had been treated with neoadjuvant chemotherapy (NAC). We also analyzed HMGB1 and calreticulin expression in breast cancer cell lines treated with chemotherapeutic drugs. As a result, both HMGB1 and calreticulin expression levels were significantly upregulated after NAC in both breast cancer and ESCC tissues. However, no significant correlation was observed between HMGB1 expression and pathological response after NAC or between HMGB1 expression and patient survival. Furthermore, although overall survival in the high infiltration group of CD8-positive T cells was significantly superior to that in the low infiltration group in breast cancer patients, there were no correlations between the number of CD8-positive T cells and HMGB1 or calreticulin expression levels. In addition, chemotherapeutic drugs induced upregulation of HMGB1 and calreticulin in all tested cell lines. Our findings indicate that chemotherapy alone can significantly induce ICD regardless of the degree of pathological response after chemotherapy.

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Figures

Figure 1.
Figure 1.
Representative immunostainings for HMGB1 and calreticulin in breast cancer (A) and ESCC (B) cases using anti-HMGB1, anti-calreticulin mAbs. The grade of HMGB1 and calreticulin expression was scored as 0 (0–10% positive), 1+ (>10–30% positive), 2+ (>30–80% positive), or 3+ (>80% positive) based on the tumor cells. Sections in individual sample were observed at a magnification of ×400. HMGB1, high-mobility group box 1 protein; ESCC, esophageal squamous cell carcinoma.
Figure 2.
Figure 2.
Representative immunostainings of HMGB1 and calreticulin before and after NAC in breast cancer (A) and ESCC (B) cases. Sections in individual samples were observed at a magnification of ×400. HMGB1, high-mobility group box 1 protein; NAC, neoadjuvant chemotherapy; ESCC, esophageal squamous cell carcinoma.
Figure 3.
Figure 3.
Summarized data for semi-quantitative evaluation of HMGB1 and calreticulin expression before and after NAC in breast cancer and ESCC cases. *p<0.05, **p<0.001. HMGB1, high-mobility group box 1 protein; NAC, neoadjuvant chemotherapy; ESCC, esophageal squamous cell carcinoma.
Figure 4.
Figure 4.
Pathological response and HMGB1/calreticulin expression as related to clinical outcomes in breast cancer patients. (A) Correlation of the pathological response to overall survival. (B) Correlation of the HMGB1 expression before NAC (left graph) and after NAC (right graph) to overall survival. (C) Correlation of calreticulin expression before NAC (left graph) and after NAC (right graph) to overall survival. *p<0.05. HMGB1, high-mobility group box 1 protein; NAC, neoadjuvant chemotherapy. n.s., not significant.
Figure 5.
Figure 5.
Correlation of the number of CD8+ T cells before and after NAC with the HMGB1 and calreticulin score, and patient survival in breast cancer patients. (A) Evaluation of the number of CD8+ T cells before and after NAC. Representative IHC of CD8 is shown. (B) Evaluation of the number of CD8+ T cells before and after NAC in pathological response. (C) Correlation of the HMGB1/calreticulin score with the number of CD8+ T cells before and after NAC. (D) Correlation of the degree of infiltrating CD8+ T cells before and after NAC with clinical outcomes. *p<0.05. HMGB1, high-mobility group box 1 protein; NAC, neoadjuvant chemotherapy. n.s. not significant.
Figure 6.
Figure 6.
In vitro treatment of breast cancer cell lines with chemotherapeutic drugs. (A) The proportion of dying cells [Annexin V(+) or 7-AAD(+)] was analyzed by flow cytometry. (B) HMGB1 in the supernatant after chemotherapy was evaluated by ELISA. (C) Calreticulin expressed on the cell surface after chemotherapy was evaluated by flow cytometry. Representative histograms for these cell lines are shown. *p<0.05, **p<0.001. PTX, paclitaxel; DXR, doxorubicin; HMGB1, high-mobility group box 1 protein. n.s., not significant.
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