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Fibroblast activation protein (FAP) generally shows low or undetectable expression in most normal tissues but is highly expressed in fibroblasts in almost all carcinomas. FAP is one of the potential molecules to detect activated fibroblasts and has multiple roles in tumour progression. We generated transgenic mice that specifically expressed tdTomato along with FAP promoter activity. Coculturing a mouse gastric cancer cell line and FAP-tdTomato transgenic mouse-derived fibroblasts showed that tdTomato expression was elevated in the cocultured fibroblasts. Moreover, stomach wall transplanted tumours in mice also showed FAP-tdTomato expression in fibroblasts of the stomach and each metastatic legion. These results indicated that FAP-tdTomato expression in fibroblasts was elevated by stimulation through the interaction with cancer cells. Functionally, collagen production was increased in FAP/tdTomato-positive fibroblasts cocultured with mouse cancer cells. These FAP-tdTomato transgenic mice have the potential to be used to investigate real-time FAP dynamics and the importance of FAP expression in tumour development.

Excess stroma and cancer-associated fibroblasts (CAF) enhance cancer progression and facilitate immune evasion. Insights into the mechanisms by which the stroma manipulates the immune microenvironment could help improve cancer treatment. Here, we aimed to elucidate potential approaches for stromal reprogramming and improved cancer immunotherapy. Platelet-derived growth factor C (PDGFC) and D expression were significantly associated with a poor prognosis in patients with gastric cancer, and PDGF receptor beta (PDGFRβ) was predominantly expressed in diffuse-type gastric cancer stroma. CAFs stimulated with PDGFs exhibited markedly increased expression of CXCL1, CXCL3, CXCL5, and CXCL8, which are involved in polymorphonuclear myeloid-derived suppressor cell (PMN-MDSC) recruitment. Fibrotic gastric cancer xenograft tumors exhibited increased PMN-MDSC accumulation and decreased lymphocyte infiltration, as well as resistance to anti–PD-1. Single-cell RNA sequencing and spatial transcriptomics revealed that PDGFRα/β blockade reversed the immunosuppressive microenvironment through stromal modification. Finally, combining PDGFRα/β blockade and anti–PD-1 treatment synergistically suppressed the growth of fibrotic tumors. These findings highlight the impact of stromal reprogramming on immune reactivation and the potential for combined immunotherapy for patients with fibrotic cancer.Significance:Stromal targeting with PDGFRα/β dual blockade reverses the immunosuppressive microenvironment and enhances the efficacy of immune checkpoint inhibitors in fibrotic cancer.See related commentary by Tauriello, p. 655

Supplementary Figure8

Excess stroma and cancer-associated fibroblasts (CAF) enhance cancer progression and facilitate immune evasion. Insights into the mechanisms by which the stroma manipulates the immune microenvironment could help improve cancer treatment. Here, we aimed to elucidate potential approaches for stromal reprogramming and improved cancer immunotherapy. Platelet-derived growth factor C (PDGFC) and D expression were significantly associated with a poor prognosis in patients with gastric cancer, and PDGF receptor beta (PDGFRβ) was predominantly expressed in diffuse-type gastric cancer stroma. CAFs stimulated with PDGFs exhibited markedly increased expression of CXCL1, CXCL3, CXCL5, and CXCL8, which are involved in polymorphonuclear myeloid-derived suppressor cell (PMN-MDSC) recruitment. Fibrotic gastric cancer xenograft tumors exhibited increased PMN-MDSC accumulation and decreased lymphocyte infiltration, as well as resistance to anti–PD-1. Single-cell RNA sequencing and spatial transcriptomics revealed that PDGFRα/β blockade reversed the immunosuppressive microenvironment through stromal modification. Finally, combining PDGFRα/β blockade and anti–PD-1 treatment synergistically suppressed the growth of fibrotic tumors. These findings highlight the impact of stromal reprogramming on immune reactivation and the potential for combined immunotherapy for patients with fibrotic cancer.Significance:Stromal targeting with PDGFRα/β dual blockade reverses the immunosuppressive microenvironment and enhances the efficacy of immune checkpoint inhibitors in fibrotic cancer.See related commentary by Tauriello, p. 655

Supplementary Information

Epithelial-mesenchymal transition (EMT) is one of essential processes in cancer progression. Plasticity between epithelial and mesenchymal status is referred as epithelial-mesenchymal plasticity (EMP). The significance of spontaneous change of EMP is under investigation in the field of EMT. To date, spontaneous EMP is reported in breast and prostate cancer, which leads to the acquisition of stem-cell properties and chemoresistance. On the other hand, the presence of spontaneous EMP is still not examined in other cancer including esophageal cancer. In the present study, we tackled the question of whether esophageal cancer have the potency of spontaneous EMP. We screened eleven esophageal squamous cancer cell (ESCC) cell lines with CD44 isoform expression, which is known to reflect epithelial and mesenchymal status. Interestingly, KYSE520 was found to comprise heterogenous populations consisting of CD44v+ and CD44v- subpopulations. CD44v+ and CD44v- cells showed the expression of epithelial and mesenchymal markers, respectively. Single-cell sorting of CD44v+ and CD44v- cells revealed both cells gave rise to cell populations consisting of CD44v+ and CD44v- cells, indicating CD44v+ epithelial-like and CD44v- mesenchymal-like cells can generate counterparts, respectively. This EMP was maintained in next generation. Further, we found the generation of counterparts between CD44v+ and CD44v- cells in vivo. Epithelial splicing regulatory protein 1 (ESRP1) is reported as the regulator of CD44 mRNA splicing. The role of ESRP1 in EMP is not well understood in esophageal cancer. The inhibition of ESRP1 shifted CD44v+ to CD44v- cells in KYSE520. However, the expression of epithelial-mesenchymal transition (EMT)-related markers or transcriptional factors were almost not affected, suggesting ESRP1 functions downstream of EMP. Our results firstly revealed the presence of spontaneous EMP in esophageal cancer and give a new insight in the field of EMT. Citation Format: Kenji Tsuchihashi, Yuki Hirata, Juntaro Yamasaki, Kentaro Suina, Kenro Tanoue, Toshifumi Yae, Kenta Masuda, Eishi Baba, Koichi Akashi, Yuko Kitagawa, Hideyuki Saya, Osamu Nagano. Esophageal cancer shows spontaneous plasticity between epithelial and mesenchymal status [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1227.

Purpose Angiopoietin-like 4 (ANGPTL4) was recently shown to be associated with cancer progression but little is known about its contribution to cancer metabolism. The purpose of this study was to elucidate the role of ANGPTL4 in glucose metabolism in colorectal cancer (CRC). Methods Immunohistochemical staining of CRC specimens classified 84 patients into two groups according to ANGPTL4 expression. Clinicopathological characteristics, gene mutation status obtained by next-generation sequencing, and fluorodeoxyglucose (FDG) uptake measured by positron emission tomography/computed tomography (PET/CT) were compared between the two groups. Furthermore, the impact of ANGPTL4 expression on cancer metabolism was investigated by a subcutaneous xenograft mouse model using the ANGPTL4 knockout CRC cell line and glucose transporter (GLUT) expression was evaluated. Results There were significantly more cases of T3/4 tumours (94.3% vs. 57.1%, P < 0.001) and perineural invasion (42.9% vs. 22.4%, P = 0.046) in the ANGPTL4-high group than in the low group. Genetic exploration revealed a higher frequency of KRAS mutation (54.3% vs. 22.4%, P = 0.003) in the ANGPTL4-high tumours. All the FDG uptake parameters were significantly higher in ANGPTL4-high tumours. In vivo analysis showed a significant reduction in tumor size due to ANGPTL4 knockout with lower expression of GLUT1 and GLUT3, and suppression of AKT phosphorylation. Conclusion ANGPTL4 regulates the expression of GLUTs by activating the PI3K-AKT pathway and thereby promoting glucose metabolism in CRC. These findings establish a new functional role of ANGPTL4 in cancer progression and lay the foundation for developing a novel therapeutic target.

Autocrine and paracrine factors, including glutamate and epidermal growth factor (EGF), are potent inducers of brain tumor cell invasion, a pathological hallmark of malignant gliomas. System xc(-) consists of xCT and CD98hc subunits and functions as a plasma membrane antiporter for the uptake of extracellular cystine in exchange for intracellular glutamate. We previously showed that the EGF receptor (EGFR) interacts with xCT and thereby promotes the activity of system xc(-) in a kinase-independent manner, resulting in enhanced glutamate release in glioma cells. However, the molecular mechanism underlying EGFR-mediated glioma progression in a glutamate-rich microenvironment has remained unclear. Here we show that the GluN2B subunit of the N-methyl-d-aspartate-sensitive glutamate receptor (NMDAR) is a substrate of EGFR in glioma cells. In response to EGF stimulation, EGFR phosphorylated the COOH-terminal domain of GluN2B and thereby enhanced glutamate-NMDAR signaling and consequent cell migration in EGFR-overexpressing glioma cells. Treatment with the NMDAR inhibitor MK-801 or the system xc(-) inhibitor sulfasalazine suppressed EGF-elicited glioma cell migration. The administration of sulfasalazine and MK-801 also synergistically suppressed the growth of subcutaneous tumors formed by EGFR-overexpressing glioma cells. Furthermore, shRNA-mediated knockdown of xCT and GluN2B cooperatively prolonged the survival of mice injected intracerebrally with such glioma cells. Our findings thus establish a central role for EGFR in the signaling crosstalk between xCT and GluN2B-containing NMDAR in glioma cells.

The cystine-glutamate antiporter subunit xCT suppresses iron-dependent oxidative cell death (ferroptosis) and is therefore a promising target for cancer treatment. Given that cancer cells often show resistance to xCT inhibition resulting in glutathione (GSH) deficiency, however, we here performed a synthetic lethal screen of a drug library to identify agents that sensitize the GSH deficiency-resistant cancer cells to the xCT inhibitor sulfasalazine. This screen identified the oral anesthetic dyclonine which has been recently reported to act as a covalent inhibitor for aldehyde dehydrogenases (ALDHs). Treatment with dyclonine induced intracellular accumulation of the toxic aldehyde 4-hydroxynonenal in a cooperative manner with sulfasalazine. Sulfasalazine-resistant head and neck squamous cell carcinoma (HNSCC) cells were found to highly express ALDH3A1 and knockdown of ALDH3A1 rendered these cells sensitive to sulfasalazine. The combination of dyclonine and sulfasalazine cooperatively suppressed the growth of highly ALDH3A1-expressing HNSCC or gastric tumors that were resistant to sulfasalazine monotherapy. Our findings establish a rationale for application of dyclonine as a sensitizer to xCT-targeted cancer therapy.

Targeting the function of membrane transporters in cancer stemlike cells is a potential new therapeutic approach. Cystine‐glutamate antiporter xCT expressed in CD44 variant (CD44v)‐expressing cancer cells contributes to the resistance to oxidative stress as well as cancer therapy through promoting glutathione (GSH)‐mediated antioxidant defense. Amino acid transport by xCT might, thus, be a promising target for cancer treatment, whereas the determination factors for cancer cell sensitivity to xCT‐targeted therapy remain unclear. Here, we demonstrate that high expression of xCT and glutamine transporter ASCT2 is correlated with undifferentiated status and diminished along with cell differentiation in head and neck squamous cell carcinoma (HNSCC). The cytotoxicity of the xCT inhibitor sulfasalazine relies on ASCT2‐dependent glutamine uptake and glutamate dehydrogenase (GLUD)‐mediated α‐ketoglutarate (α‐KG) production. Metabolome analysis revealed that sulfasalazine treatment triggers the increase of glutamate‐derived tricarboxylic acid cycle intermediate α‐KG, in addition to the decrease of cysteine and GSH content. Furthermore, ablation of GLUD markedly reduced the sulfasalazine cytotoxicity in CD44v‐expressing stemlike HNSCC cells. Thus, xCT inhibition by sulfasalazine leads to the impairment of GSH synthesis and enhancement of mitochondrial metabolism, leading to reactive oxygen species (ROS) generation and, thereby, triggers oxidative damage. Our findings establish a rationale for the use of glutamine metabolism (glutaminolysis)‐related genes, including ASCT2 and GLUD, as biomarkers to predict the efficacy of xCT‐targeted therapy for heterogeneous HNSCC tumors.

Background: Head and Neck Squamous Cell Carcinoma (HNSCC) is the sixth most common cancer in the world. Despite the development of various treatments, the five-year survival rate for HNSCC is about 50-60% and has not been improved in recent years. Immune checkpoint inhibitors are promising, but the effect of those is limited. The development of new reagents having different mechanisms against tumor progression is still needed. From the result of drug screening, Pimozide (PMZ), an antipsychotic drug used for the treatment of schizophrenia and chronic psychosis, demonstrated a strong antitumor ability to HNSCC cells expressing high level of CD44v. In this study, we examined the combined effects of PMZ and paclitaxel (PTX) for HNSCC cells. Methods: Using HNSCC cell lines (FaDu, Detroit562, HSC-2, OSC-19), the antitumor effects of PTX, PMZ, and the combination of PTX and PMZ were evaluated by MTS assay. Moreover, the reactive oxygen species (ROS) expression level under each drug administration was measured by flow cytometry. Moreover, PTX, PMZ (0.45mg/kg), PMZ (1mg/kg), PTX+PMZ were administered to the xenograft model by OSC-19. The tumor was resected after those treatments. The expression of CD44v, ALDH3A1, and NRF2 was evaluated by immunohistochemistry (IHC) of FFPE tissues from those extracted tumors. Results: PMZ single-agent inhibited tumor growth in various tumor cell lines, and the antitumor effect was enhanced by concomitant use of PTX. PMZ, which significantly increased ROS of tumor cells, was more effective than PTX in OSC-19, which was a chemoresistant cell line expressing high CD44v. Xenograft model exhibited that tumor growth was suppressed in PTX+PMZ group than in PTX and PMZ single-agent group. IHC staining revealed that PMZ administration restrained CD44v expression, and ALDH3A1 expression markedly decreased in PMZ treated tumors, but not in PTX treated tumors. Conclusions: PMZ dramatically induced ROS production in tumor cells. These data suggest that aldehyde produced by ROS might suppress HNSCC tumor growth. The results from this study indicated that PMZ could be a novel therapeutic reagent for HNSCC in combination with existing anticancer drugs. Citation Format: Shintaro Nakamura, Hiroyuki Ozawa, Juntaro Yamasaki, Shogo Okazaki, Momoko Yoshikawa, Tomoya Soma, Seiji Asoda, Osamu Nagano, Mariko Sekimizu, Shin Saito, Keisuke Yoshihama, Takuya Mikoshiba, Kaoru Ogawa, Hideyuki Saya. Pimozide combined with paclitaxel demonstrates a significant antitumor effect in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3579.

Small Cell Lung Cancer (SCLC) which accounts for 15 % of primary lung cancers is known to grow fast and metastasize easily to whole body at early stages. However, the standard care of SCLC has not been changed for over 10 years and effective treatment remains to be developed. System xc(-) comprises xCT (SLC7A11) and CD98hc subunits and is a major plasma membrane antiporter responsible for the cellular uptake of cystine in exchange for intracellular glutamate. Recently, we studied the impact of xCT inhibitor sulfasalazine (SSZ) in various cancers and found that SCLC cells manifest low level of xCT and are highly sensitive to SSZ treatment compared with non-small cell lines. Furthermore, we found that xCT expression in SCLC cells is regulated by the cellular density and the tumorsphere culture which is known to enhance the cancer stem cell properties also enhances the xCT expression in these cells. To examine the functional relevance of xCT expression to cancer stem-like properties in SCLC cells, we established the stably xCT-expressing SBC-5 cells (SBC5-xCT). Similar to other SCLC cell lines, parental SBC5 cells were not able to proliferate from single-cell level, however, SBC5-xCT cells were found to possess an ability to proliferate from single-cell level. Furthermore, SBC5-xCT cells manifested higher resistance to an anticancer agent cisplatin and higher ability of tumor-initiation than parental SBC5 cells. Our findings establish a new functional role for xCT in the promotion of cancer stem-like properties in SCLC cells. Citation Format: Shohei Kamenori, Kentaro Suina, Juntaro Yamasaki, Subaru Shintani, Yuji Otsuki, Yuki Hirata, Shogo Okazaki, Kenji Tsuchihashi, Oltea Sampetrean, Yoichiro Mitsuishi, Fumiyuki Takahashi, Kazuhisa Takahashi, Hideyuki Saya, Osamu Nagano. SLC7A11 expression confers cancer stem-like properties in small cell lung cancer cells [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 4639.