Catumaxomab with Activated T-cells Efficiently Lyses Chemoresistant EpCAM-positive Triple-negative Breast Cancer Cell Lines.

BACKGROUND/AIM: Epithelial cell adhesion molecule (EpCAM) is expressed in various types of cancer, including breast cancer, and is correlated with metastasis, invasion, therapeutic resistance and prognosis. Moreover, several cell surface markers, such as CD44 and EpCAM, are molecular targets on cancer stem-like cells of breast cancer. The aim of this study was to investigate whether catumaxomab, a clinical-grade bispecific antibody that binds to both EpCAM on tumor cells and CD3 on T-cells, combined with activated T-cells can eliminate chemoresistant triple-negative breast cancer (TNBC) cells in vitro. MATERIALS AND METHODS: First, a cell line (MUK-BC1) was established from human breast carcinoma cells derived from a patient with chemoresistant and disseminated breast cancer. These EpCAM-positive TNBC cells were almost completely resistant to various drug-mediated cytotoxicities up to a concentration of 10 µg/ml. RESULTS: Pre-treatment with catumaxomab and subsequent addition of interleukin-2/OKT3-activated autologous T-cells eliminated EpCAM-positive TNBC cells. CONCLUSION: Catumaxomab combined with activated T-cells may be a potent therapeutic modality to overcome chemoresistant EpCAM-positive TNBC cells.

[Development of Holistic Cancer Treatment Centering Cancer Patients - From the Standpoint of Hypoxia and Hedgehog Signaling].

Recently, hypoxia that is one of cancer microenvironments, takes much attention. Because circumstance that we usually perform experiment is 20% O2 condition, it is likely that different signaling pathways may be activated in vivo cancer. We focused Hedgehog(Hh)signaling as one of activated pathways under hypoxia. It has been shown that Hh signaling is activated under hypoxia, followed by inducing malignant phenotypes in pancreatic cancer. Therefore, Hh signaling inhibitor should elicit anti-tumor effect. However, if we consider "whole-person therapy" we should confirm how Hh signaling affects the function of immune cells. In the present study, we describe hypoxia/Hh signaling/functions of cancer cells and immune cells focusing our previous results.

Hedgehog signaling regulates PDL-1 expression in cancer cells to induce anti-tumor activity by activated lymphocytes.

We investigated whether hypoxia-induced activation of Hh signaling contributes to PDL-1 expression in cancer and whether it affects the anti-tumor function of activated lymphocytes. Hypoxia augmented PDL-1 expression and inhibition of Hh signaling reduced PDL-1 expression under hypoxia. When activated lymphocytes were cocultured with cancers treated with a Hh inhibitor, activated lymphocyte cell numbers increased under hypoxia. In contrast, this increase was abrogated when cancer cells were treated with a PDL-1 neutralizing antibody. These results suggest that Hh signaling is one of regulatory pathways of PDL-1 expression under hypoxia and that inhibiting Hh signaling may induce lymphocyte anti-tumor activity.

Protein-bound Polysaccharide-K Inhibits Hedgehog Signaling Through Down-regulation of MAML3 and RBPJ Transcription Under Hypoxia, Suppressing the Malignant Phenotype in Pancreatic Cancer.

Hedgehog signaling is activated in pancreatic cancer and could be a therapeutic target. We previously demonstrated that recombination signal binding protein for immunoglobulin-kappa-J region (RBPJ) and mastermind-like 3 (MAML3) contribute to the hypoxia-induced up-regulation of Smoothened (SMO) transcription. We have also shown that protein-bound polysaccharide-K (PSK) could be effective for refractory pancreatic cancer that down-regulates SMO transcription under hypoxia. In this study, we evaluated whether the anticancer mechanism of PSK involves inhibiting RBPJ and MAML3 expression under hypoxia. PSK reduced SMO, MAML3 and RBPJ expression in pancreatic cancer cells under hypoxia. PSK also blocked RBPJ-induced invasiveness under hypoxia by inhibiting matrix metalloproteinase expression. Lastly, we showed that PSK attenuated RBPJ-induced proliferation both in vitro and in vivo. These results suggest that PSK suppresses Hedgehog signaling through down-regulation of MAML3 and RBPJ transcription under hypoxia, inhibiting the induction of a malignant phenotype in pancreatic cancer. Our results may lead to development of new treatments for refractory pancreatic cancer using PSK as a Hedgehog inhibitor.

Nuclear expression of Y-box binding protein-1 is associated with poor prognosis in patients with pancreatic cancer and its knockdown inhibits tumor growth and metastasis in mice tumor models.

The objective of this study was to examine the implication of Y-box-binding protein-1 (YB-1) for the aggressive phenotypes, prognosis and therapeutic target in pancreatic ductal adenocarcinoma (PDAC). YB-1 expression in PDAC, pancreatic intraepithelial neoplasia (PanIN) and normal pancreas specimens was evaluated by immunohistochemistry, and its correlation with clinicopathological features was assessed in patients with PDAC. The effects of YB-1 on proliferation, invasion and expressions of cell cycle-related proteins and matrix metalloproteinases (MMPs) were analyzed by WST-8, cell cycle and Matrigel invasion assays, Western blotting and quantitative RT-PCR in PDAC cells transfected with YB-1-siRNAs. To verify the significance of YB-1 for tumor progression in vivo, the growth and metastasis were monitored after intrasplenic implantation of ex vivo YB-1 siRNA-transfected PDAC cells, and YB-1-targeting antisense oligonucleotides were intravenously administered in nude mice harboring subcutaneous tumor. The intensity of YB-1 expression and positivity of nuclear YB-1 expression were higher in PDAC than PanIN and normal pancreatic tissues. Nuclear YB-1 expression was significantly associated with dedifferentiation, lymphatic/venous invasion and unfavorable prognosis. YB-1 knockdown inhibited cell proliferation via cell cycle arrest by S-phase kinase-associated protein 2 downregulation and consequent p27 accumulation, and decreased the invasion due to downregulated membranous-type 2 MMP expression in PDAC cells. Tumor growth and liver metastasis formation were significantly suppressed in nude mice after implantation of YB-1-silenced PDAC cells, and the YB-1 targeting antisense oligonucleotide significantly inhibited the growth of subcutaneous tumors. In conclusion, YB-1 may be involved in aggressive natures of PDAC and a promising therapeutic target.

CD24 suppresses malignant phenotype by downregulation of SHH transcription through STAT1 inhibition in breast cancer cells.

Hedgehog (Hh) signaling has been found to be activated in breast cancer stem cells (BCSCs). However, the precise role of the BCSCs marker, CD24, remains unclear. Here, we describe a relationship between CD24 and Sonic Hedgehog (SHH), and reveal a role for this relationship in the induction of a malignant phenotype of breast cancer. CD24 siRNA-transfected breast cancer cells (BCCs) demonstrated higher expression of SHH and GLI1, increased anchorage-independent proliferation, and enhanced invasiveness and superior tumorigenicity compared with control. Conversely, CD24 forced-expressing BCCs possessed decreased SHH and GLI1 expression, anchorage-independent proliferation, and invasiveness. Suppression of SHH decreased invasiveness through inhibition of matrix metalloproteinase (MMP)-2 expression, GLI1 expression, anchorage-independent proliferation, tumorigenicity, and tumor volume in vivo in CD24 siRNA transfected BCCs. DNA microarray analysis identified STAT1 as a relationship between CD24 and SHH. CD24 siRNA-transfected BCCs with concurrent STAT1 inhibition exhibited decreased SHH expression, invasiveness, anchorage-independent proliferation, tumorigenicity, and tumor volume in vivo. These results suggest that CD24 suppresses development of a malignant phenotype by down-regulating SHH transcription through STAT1 inhibition. CD24 gene transfer or STAT1 inhibition may represent new effective therapeutic strategies to target refractory breast cancer.

Hypoxia but not normoxia promotes Smoothened transcription through upregulation of RBPJ and Mastermind-like 3 in pancreatic cancer.

We previously demonstrated that Hedgehog (Hh) signaling is activated under hypoxia through upregulation of transcription of Smoothened (SMO) gene. However, the mechanism of hypoxia-induced activation of SMO transcription remains unclear. In the analysis of altered expressions of genes related to Hh signaling between under normoxia and hypoxia by DNA microarray analysis, we picked up 2 genes, a transcriptional regulator, recombination signal binding protein for immunoglobulin-kappa-J region (RBPJ) and a transcriptional co-activator, Mastermind-like 3 (MAML3). Expressions of SMO, MAML3 and RBPJ were increased under hypoxia in pancreatic ductal adenocarcinoma cells (PDAC). RBPJ and MAML3 inhibition under hypoxia led to decreased SMO and GLI1 expressions, whereas SMO expression in MAML3-inhibited and RBPJ-inhibited cells under normoxia showed no change. However, overexpression of RBPJ under normoxia led to increased SMO expression. Additionally, cells knocked down for MAML3 and RBPJ inhibition under hypoxia showed decreased invasiveness through matrix metalloproteinase-2 suppression and decreased proliferation. Xenograft mouse models showed that MAML3 and RBPJ knockdown inhibited tumorigenicity and tumor volume. Our results suggest that hypoxia promotes SMO transcription through upregulation of MAML3 and RBPJ to induce proliferation, invasiveness and tumorigenesis in pancreatic cancer.

New Approach to Complete Video-assisted Thoracoscopic Lobectomy in T2 and T3 Non-Small Cell Lung Cancer.

Complete video-assisted thoracoscopic surgery (c-VATS) for lung cancer is minimally invasive because of the small incision required. c-VATS has recently become a standard procedure for treatment of stage IA/IB lung cancer. However, a long thoracic incision or extensive costal rib resection is required in patients with large lung tumors. We herein introduce an improved VATS lobectomy procedure for patients with T2 and T3 lung cancer. In this technique, resected tissue is removed through a small upper abdominal midline incision below the xiphoid through the retrosternal-extraperitoneal pathway. Five patients who underwent this new procedure were compared against 10 control patients who underwent hybrid VATS lobectomy. Significantly fewer patients who underwent c-VATS lobectomy complained of severe postoperative pain; however, there was no significant difference in the postoperative hospital stay between the two groups. The present study demonstrates that c-VATS lobectomy can be performed with minimal operative pain and without need for a long thoracic incision or extensive rib resection, even in patients with large lung tumors (T2 and T3). These results suggest that the indications for c-VATS lobectomy in patients with T2 and T3 non-small cell lung cancer can be expanded by implementation of our approach, which involves removal of the freed lobe through an abdominal incision.

Surgical treatment to aid patients with colorectal perforation.

BACKGROUND: When a patient with colorectal perforation is treated, there is no adequate information about what the best procedure for emergent operation is. In the present study, we examined the clinical features in hemodialysis (HD) and non-HD patients with colorectal perforations. PATIENTS AND METHODS: Forty-four patients (8 HD and 36 non-HD patients) who underwent surgery for colorectal perforation at the Fukuoka Red Cross Hospital were reviewed and analyzed. RESULTS: Poor prognostic factors for patients with colorectal perforation were high age, HD, idiopathic perforation, postoperative complication and Hartmann's operation procedure. Good prognostic factors were diverticulum perforation and operation with anastomosis. In the analysis between HD and non-HD patients, clinical characteristics in HD patients with colorectal perforation were advanced age and rectal perforation. CONCLUSION: High mortality rate of HD patients with colorectal perforation may be due to high age and rectal perforation in which cases it is technically difficult to perform operation. In case of emergent operation of colorectal perforation, the Hartmann's procedure may not be recommended.

Random migration contributes to cytotoxicity of activated CD8+ T-lymphocytes but not NK cells.

Activated lymphocytes have the ability to undergo non-directional cell movement known as random migration, although the biological role for this remains unclear. Herein, we investigated how random migration affects cytotoxicity of activated lymphocytes using time-lapse imaging analysis. The kinetics of random migration paralleled cytotoxicity in activated lymphocytes. Sphingosine-1-phosphate (S1P) and its receptor-1 (S1PR1) play an important role in lymphocyte migration. Phosphorylated FTY720 (FTYP), a structural analog of S1P, significantly inhibited random migration and cytotoxicity of activated CD3(+)NKG2D(+)CD8(+) T-lymphocytes but not CD3(-)NKG2D(+)CD56(+) natural killer (NK) cells. In a mouse xenograft model, FTYP-treated activated lymphocytes exhibited lower cytotoxicity and less tumor infiltration for activated CD3(+)NKG2D(+) T-lymphocytes but not CD3(-)NKG2D(+) NK cells. These results suggest that random migration contributes to the cytotoxicity of activated CD8(+) T-cells but not of NK cells.

An epithelial cell adhesion molecule- and CD3-bispecific antibody plus activated T-cells can eradicate chemoresistant cancer stem-like pancreatic carcinoma cells in vitro.

Cancer stem-like properties of various types of cancer, including pancreatic cancer, one of the most aggressive types, correlate with metastasis, invasion, and therapeutic resistance. More importantly, chemoresistance in cancer stem-like cells (CSLCs) is a critical problem for eradication of pancreatic cancer. Several cell surface markers, such as CD44 and epithelial cell adhesion molecule (EpCAM), are molecular targets on CSLCs of pancreatic carcinoma. In this study, we investigated whether catumaxomab, a clinical-grade bi-specific antibody that binds to both EpCAM on tumor cells and CD3 on T-cells, combined with activated T-cells can eliminate chemoresistant pancreatic CSLCs in vitro. Firstly, we established a CSLC line (MU-PK1) from human pancreatic carcinoma cells derived from a patient with chemoresistant and disseminated pancreatic cancer. These CSLCs were almost completely resistant to gemcitabine-mediated cytotoxicity up to a concentration of 10 µg/ml. The cells expressed high levels of CSLC markers (CD44 and EpCAM) and had significantly higher capacities for sphere formation, invasion, and aldehyde dehydrogenase-1 expression, which are associated with cancer stemness properties. We found that pre-treatment with catumaxomab and subsequent addition of interleukin-2/OKT3 activated autologous T-cells eliminated CSLCs during a short incubation period. Moreover, when MU-PK1 cells were cultured under hypoxic conditions, the CSLCs became more aggressive. However, the combination of cytokine-activated killer T-cells with catumaxomab successfully lysed almost all these cells. In conclusion, catumaxomab combined with activated T-cells may be a potent therapeutic modality to eradicate chemoresistant pancreatic CSLCs.

NKG2D-directed cytokine-activated killer lymphocyte therapy combined with gemcitabine for patients with chemoresistant metastatic solid tumors.

Natural-killer group 2, member D (NKG2D) is an activating receptor found on activated natural killer cells and on activated T-cells, here termed cytokine-activated killer (CAK) cells. NKG2D ligands are expressed on various human cancer types. Gemcitabine is an anticancer drug which is a less immune-destructive agent than others. Herein, we investigated the clinical efficacy and the underlying mechanisms of a combination of CAK cell infusion therapy and gemcitabine. Twenty-three patients with disseminated carcinomas were treated with chemo-immunotherapy consisting of CAK cell infusion therapy following gemcitabine treatment. To investigate the underlying mechanisms by which CAK cells synergize with gemcitabine, we used enzyme-linked immunosorbent assay, Real-time reverse transcription polymerase chain reaction assay, calcein-release assay, and adherent target detachment assay. Using these assays we determined the NKG2D ligands such as major histocompatibility complex-class I-related chain (MIC)A/B expression in carcinoma cells and the level of cellular cytotoxicity generated by treatment with gemcitabine with/without CAK cells. The tumor responses differed among the patients (n=23). In vitro experiments revealed that MICA/B protein and mRNA expression were up-regulated in several carcinoma cell lines after gemcitabine treatment. Pre-treatment with gemcitabine and subsequent exposure to CAK cells induced greater cytotoxicity than either treatment alone. Inclusion of soluble MICB in CAK cell-mediated cytotoxicity assay significantly reduced cytotoxicity. Our clinical results of gemcitabine-CAK combinatorial therapy demonstrated long-term stable disease despite chemoresistance. In conclusion, the combination of gemcitabine and CAK cells may have clinical therapeutic significance for pancreatic, hepato-biliary tract, and urothelial tract cancer. Our study shows that combining CAK therapy with gemcitabine can lead to successful treatment of metastatic cancer.

Prognostic significance of urine N1, N12-diacetylspermine in patients with non-small cell lung cancer.

BACKGROUND: Recently N(1),N(12)-diacetylspermine, a diacetylated polyamine derivative, was recognized as a tumor marker in patients with several kinds of cancers. However, the significance of its levels in urine as a prognostic factor has not been elucidated. In the present study, we examined whether the urine N(1),N(12)-diacetylspermine levels can be used as a prognostic factor in patients with NSCLC. PATIENTS AND METHODS: Urine samples from 251 patients with NSCLC were collected prior to surgery and the urinary N(1),N(12)-diacetylspermine concentration was measured. Thereafter, all 251 patients underwent curative surgery and the analysis of prognosis was performed for over 10 years. Out of the 251 patients, 91 had recurrent disease. The significance of the urinary N(1),N(12)-diacetylspermine level as a prognostic factor among all 251 patients and among the 91 patients with recurrence was evaluated. RESULTS: Univariate analysis of all 251 patients showed that the level of urinary N(1),N(12)-diacetylspermine was a significant prognostic factor for disease-free survival and overall survival; however, multivariate analysis showed it had no significance. Conversely, the univariate and multivariate analyses of post-recurrent survival of the 91 patients with recurrence showed that urinary N(1),N(12)-diacetylspermine was an independent prognostic factor for post-recurrent survival. CONCLUSION: Patients with recurrence with positive urinary N(1),N(12)-diacetylspermine should undergo more intensive care and determination of urinary N(1),N(12)-diacetylspermine may contribute to improvement of prognosis of NSCLC.

Hedgehog signaling pathway as a new therapeutic target in pancreatic cancer.

Pancreatic cancer is one of the most aggressive and difficult cancers to treat. Despite numerous research efforts, limited success has been achieved in the therapeutic management of patients with this disease. In the current review, we focus on one component of morphogenesis signaling, Hedgehog (Hh), with the aim of developing novel, effective therapies for the treatment of pancreatic cancer. Hh signaling contributes to the induction of a malignant phenotype in pancreatic cancer and is responsible for maintaining pancreatic cancer stem cells. In addition, we propose a novel concept linking Hh signaling and tumor hypoxic conditions, and discuss the effects of Hh inhibitors in clinical trials. The Hh signaling pathway may represent a potential therapeutic target for patients with refractory pancreatic cancer.

Hedgehog signaling pathway is a potential therapeutic target for gallbladder cancer.

Gallbladder cancer (GBC) is a particularly deadly type of cancer with a 5-year survival rate of only 10%. New effective therapeutic strategies are greatly needed. Recently, we have shown that Hedgehog (Hh) signaling is reactivated in various types of cancer and is a potential therapeutic target. However, little is known about the biological significance of Hh signaling in human GBC. In this study, we determined whether Hh signaling could be a therapeutic target in GBC. The Hh transcription factor Gli1 was detected in the nucleus of GBC cells but not in the nucleus of normal gallbladder cells. The expression levels of Sonic Hh (Shh) and Smoothened (Smo) in human GBC specimens (n = 37) were higher than those in normal gallbladder tissue. The addition of exogenous Shh ligand augmented the anchor-dependent and anchor-independent proliferation and invasiveness of GBC cells in vitro. In contrast, inhibiting the effector Smo decreased the anchor-dependent and anchor-independent proliferation. Furthermore, the suppression of Smo decreased GBC cell invasiveness through the inhibition of MMP-2 and MMP-9 expression and inhibited the epithelial-mesenchymal transition. In a xenograft model, tumor volume in Smo siRNA-transfected GBC cells was significantly lower than in control tumors. These results suggest that Hh signaling is elevated in GBC and may be involved in the acquisition of malignant phenotypes, and that Hh signaling may be a potential therapeutic target for GBC.

Reoxygenation from chronic hypoxia promotes metastatic processes in pancreatic cancer through the Hedgehog signaling.

Pancreatic ductal adenocarcinoma (PDAC) is among the most deadly types of malignancies because of its high ability to metastasize. PDAC is thought to be under hypoxic condition. Therefore, to investigate the mechanism of metastatic processes, chronic-hypoxia-resistant PDAC cells were newly generated under hypoxic condition for 3-6 months and reoxygenation experiments were performed using these chronic-hypoxia-resistant PDAC cells in in vivo-mimicking conditions. Proliferation, invasiveness and tumorigenicity in PDAC cells were significantly increased by reoxygenation. A Hedgehog (Hh) signaling component, Gli1, was significantly increased by reoxygenation. Gli1 knockdown inhibited reoxygenation-induced increases in proliferation and tumorigenicity and decreased invasiveness through suppression of matrix metalloproteinase (MMP) 2 and MMP9. Moreover, inhibition of Sonic Hh and Smoothened abrogated reoxygenation induced increases in proliferation and invasiveness. These results suggest that metastatic processes in PDAC are induced through activation of the Hh signaling pathway. Therefore, the Hh signaling pathway may be a therapeutic target for refractory PDAC in metastatic processes induced by reoxygenation.

NOTCH4 is a potential therapeutic target for triple-negative breast cancer.

BACKGROUND/AIM: The prognosis for triple-negative breast cancer (TNBC) is poor. In the present study, we evaluated whether NOTCH4 receptor is a potential new therapeutic target for TNBC. MATERIALS AND METHODS: In vitro proliferation and invasiveness were evaluated in TNBC cells with or without small-interfering RNA (siRNA) for NOTCH4, and with or without NOTCH4 plasmid transfection. In vivo, MDA-MB-231 cells with or without NOTCH4 siRNA were subcutaneously implanted into the flank regions of mice. The frequency of nuclear translocation of NOTCH4 was assessed by immunohistochemistry in 21 TNBC samples and 46 non-TNBC samples. RESULTS: NOTCH4 inhibition in TNBC cells reduced proliferation and invasiveness, and NOTCH4 overexpression in TNBC cells increased proliferation and invasiveness. NOTCH4 inhibition reduced tumour volume and tumourigenicity of mouse xenografts. TNBC cells had a higher frequency of nuclear translocation of NOTCH4 than other cells. CONCLUSION: NOTCH4 is a new potential therapeutic target for triple-negative breast cancer.

Tropomyosin-related kinase B inhibitor has potential for tumor regression and relapse prevention in pulmonary large cell neuroendocrine carcinoma.

Large cell neuroendocrine carcinoma (LCNEC) has an especially poor prognosis, and an effective therapeutic strategy has yet to be established. We have previously shown that the expressions of tropomyosin-related kinase B (TRKB) and brain-derived neurotrophic factor (BDNF) are high in LCNEC and that TRKB/BDNF signaling is involved in the proliferation, tumorigenesis, and invasive nature of LCNEC. Therefore, TRKB/BDNF signaling may offer a potential therapeutic target for LCNEC treatment. In the present study, we evaluated whether the TRKB tyrosine kinase inhibitor, k252a, has effects on tumor regression and relapse prevention on LCNEC, using a murine xenograft model. The LCNEC cell line and NCI-H810 cells were subcutaneously implanted into the flanks or intrathoracically injected into the bilateral pleural cavities of BALB/c nude mice. k252a significantly inhibited tumor volume, expression of matrix metalloproteinases and the formation of pleural dissemination by LCNEC. These results suggest that k252a has potential for tumor regression and relapse prevention in LCNEC. Since many patients with LCNEC suffer through the use of ineffective therapeutic strategies, a clinical trial using the TRKB inhibitor for LCNEC is urgently required.

The Hedgehog inhibitor suppresses the function of monocyte-derived dendritic cells from patients with advanced cancer under hypoxia.

Immunotherapy using monocyte derived dendritic cells (Mo-DCs) from cancer patients has been developed; however, the Mo-DCs regularly studied have been derived from non-cancer bearing donors or mice, and evaluated in normoxic conditions. In the present study, we investigated the effects of Hedgehog (Hh) inhibitors which are being developed as molecular target drugs for cancer on the functions of Mo-DCs derived from patients with advanced cancer when cultured in a tumor-like hypoxic environment. Mo-DC induction, migration, chemotaxis, phagocytosis, maturation, IL-12 p40 or p70 secretion and the allogeneic lymphocyte stimulation activity of Mo-DCs from patients with advanced cancer were all significantly inhibited by the Hh inhibitor, cyclopamine under hypoxic conditions. Our results suggest that Hh signaling plays an important role in the maintenance and function of Mo-DCs derived from patients with advanced cancer when cultured under hypoxic conditions.

The Hedgehog inhibitor cyclopamine impairs the benefits of immunotherapy with activated T and NK lymphocytes derived from patients with advanced cancer.

Hedgehog (Hh) signaling is activated in various types of cancer and contributes to the progression, proliferation, and invasiveness of cancer cells. Many Hh inhibitors are undergoing clinical trial and show promise as anticancer drugs. Hh signaling is also induced in the activated T and NK (TNK) lymphocytes that are used in immunotherapy. Activated TNK lymphocyte therapy is anticipated to work well within a tumor's hypoxic environment. However, most studies on the immunobiological functions of activated TNK lymphocytes have been conducted on healthy donor samples, under normoxic conditions. In the present study, we evaluated the effects of Hh inhibition and oxygen concentrations on the function of activated TNK lymphocytes derived from patients with advanced cancer. Proliferation, migration, surface NKG2D expression, and cytotoxicity were all significantly inhibited, and IFN-γ secretion was significantly increased upon Hh inhibitor treatment of activated TNK lymphocytes under hypoxic conditions in vitro. Tumors from mice injected with cyclopamine-treated activated TNK lymphocytes showed a significant increase in tumor size and had fewer apoptotic cells compared with the tumors in mice injected with control activated TNK lymphocytes. These results suggest that Hh signaling plays a pivotal role in activated TNK lymphocyte cell function. Combination therapy using Hh inhibitors and activated TNK lymphocytes derived from patients with advanced cancer may not be advantageous.