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SignificanceImmunosuppressive Foxp3-expressing regulatory T cells (Tregs) in tumor tissues are assumed to be clonally expanding via recognizing tumor-associated antigens. By single-cell RNA sequencing, we have searched for the molecules that are specifically expressed by such multiclonal tumor Tregs, but not by tumor-infiltrating effector T cells or natural Tregs in other tissues. The search revealed the chemokine receptor CCR8 as a candidate. Treatment of tumor-bearing mice with cell-depleting anti-CCR8 antibody indeed selectively removed multiclonal tumor Tregs without affecting effector T cells or tissue Tregs, eradicating established tumors with induction of potent tumor-specific effector/memory T cells and without activating autoimmune T cells. Thus, specific depletion of clonally expanding tumor Tregs is clinically instrumental for evoking effective tumor immunity without autoimmune adverse effects.

Background/aim Cancer stem cells (CSCs) are considered to be one of the causes of tumor recurrence after chemotherapy. The purpose of our study was to isolate CSCs from human colorectal cancer cell (CRC) lines. Materials and methods Nine CRC lines were screened based on the expression level of potential CSC markers to identify putative CSCs. Tumor formation capacity in immunodeficient mice was compared with that of their counterparts. Stemness, differentiation potency and sensitivity to 5-fluorouracil (5-FU), in vitro, were also assessed. Microarray analysis was used to characterize the features of the putative CSCs. Results COLO 201 cells were separated into two populations based on CD44 expression. CD44 positive (CD44+) cells showed significantly higher tumor formation capacity than CD44- cells in immunodeficient mice. CD44+ cells also possessed stemness properties and lower sensitivity to 5-FU in vitro. Moreover, cancer stemness and chemoresistance-related genes were highly up-regulated in CD44+ cells. Conclusion CD44+ COLO 201 cells possessed the features of CSCs; therefore, the present CSC model could serve as a valuable tool to accelerate CSC research.

Despite the success of immune checkpoint therapy, most non-small cell lung cancer (NSCLC) patients still receive conventional chemotherapy. Overcoming chemotherapy resistance by identifying specific targets should improve cancer treatment and patient survival. In this study, we examined a prospective chemotherapy-resistant subpopulation in NCI-H1975 NSCLC cells and identified a novel target, neurosecretory protein VGF (VGF). Using flow cytometry, we first analyzed the expression profile of cancer stem cell markers in 100 cancer cell lines and found that NCI-H1975 cells could be divided into three subpopulations, CD44-low (CD44-L), CD44-middle (CD44-M) and CD44-high cells based on the expression pattern of CD44. CD44-M cells account for less than 5% of the NCI-H1975 cells under normal conditions, but only this subpopulation can proliferate in vitro and in vivo after treatment with cytotoxic agents, such as paclitaxel, pemetrexed and 5-fluorouracil. Affymetrix microarray analysis revealed that VGF is specifically expressed in CD44-M cells and we hypothesized that VGF would play a critical role in chemotherapy resistance. We next employed patient-derived xenograft (PDX) models of NSCLC to confirm this hypothesis with human clinical samples. NOD/SCID mice were subcutaneously transplanted with PDX and treated with either vehicle or paclitaxel (12 mg/kg, Q3Dx3, i.v.). VGF immunohistochemistry on resected PDX revealed that the number of VGF-expressing cells increased 5.6-fold in the paclitaxel-treated group compared to the vehicle control. We also performed VGF immunohistochemistry on NSCLC surgical specimens which were obtained from 127 patients, consisting of 42 neoadjuvant chemotherapy treated cases and 85 non-treated cases. The frequency of VGF-expressing specimens was higher in neoadjuvant chemotherapy treated cases (50%) than non-treated cases (18%). These results suggest that VGF-expressing tumor subpopulation is paclitaxel-resistant even in clinical settings. We also analyzed the molecular functions of VGF. Knockdown of VGF expression by siRNAs could signifıcantly suppress paclitaxel resistance in CD44-M cells. On the other hand, overexpression of VGF could significantly induce paclitaxel resistance in CD44-L cells. These results suggest that VGF is functionally responsible for chemotherapy resistance in NSCLC patients and combined administration of an anti-VGF drug and chemotherapy could be effective for preventing cancer recurrence and prolonging cancer free survival. Citation Format: Wataru Nogami, Yumi Tona, Soichi Tofukuji, Yoshino Ishioka, Mitsunobu Matsumoto, Hajime Yamada, Kenji Kuwabara, Hidekazu Tanaka, Shigeki Adachi, Yoko Yamamoto, Ryu Kanzaki, Soichiro Funaki, Yasushi Shintani, Meinoshin Okumura, Taisei Nomura. VGF is functionally responsible for chemotherapy resistance in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 372.