Hypoxia-Inducible Factor-Dependent and Independent Mechanisms Underlying Chemoresistance of Hypoxic Cancer Cells.

In hypoxic regions of malignant solid tumors, cancer cells acquire resistance to conventional therapies, such as chemotherapy and radiotherapy, causing poor prognosis in patients with cancer. It is widely recognized that some of the key genes behind this are hypoxia-inducible transcription factors, e.g., hypoxia-inducible factor 1 (HIF-1). Since HIF-1 activity is suppressed by two representative 2-oxoglutarate-dependent dioxygenases (2-OGDDs), PHDs (prolyl-4-hydroxylases), and FIH-1 (factor inhibiting hypoxia-inducible factor 1), the inactivation of 2-OGDD has been associated with cancer therapy resistance by the activation of HIF-1. Recent studies have also revealed the importance of hypoxia-responsive mechanisms independent of HIF-1 and its isoforms (collectively, HIFs). In this article, we collate the accumulated knowledge of HIF-1-dependent and independent mechanisms responsible for resistance of hypoxic cancer cells to anticancer drugs and briefly discuss the interplay between hypoxia responses, like EMT and UPR, and chemoresistance. In addition, we introduce a novel HIF-independent mechanism, which is epigenetically mediated by an acetylated histone reader protein, ATAD2, which we recently clarified.

Hypoxia- and Postirradiation reoxygenation-induced HMHA1/ARHGAP45 expression contributes to cancer cell invasion in a HIF-dependent manner.

BACKGROUND: Cancer cells in severely hypoxic regions have been reported to invade towards tumour blood vessels after surviving radiotherapy in a postirradiation reoxygenation- and hypoxia-inducible factor (HIF)-dependent manner and cause recurrence. However, how HIF induces invasiveness of irradiated and reoxygenated cancer cells remains unclear. METHODS: Here, we identified human minor histocompatibility antigen 1 (HMHA1), which has been suggested to function in cytoskeleton dynamics and cellular motility, as a responsible factor and elucidated its mechanism of action using molecular and cellular biology techniques. RESULTS: HMHA1 expression was found to be induced at the transcription initiation level in a HIF-dependent manner under hypoxia. Boyden chamber invasion assay revealed that the induction of HMHA1 expression is required for the increase in invasion of hypoxic cancer cells. Reoxygenation treatment after ionising radiation in vitro that mimics dynamic changes of a microenvironment in hypoxic regions of tumour tissues after radiation therapy further enhanced HMHA1 expression and invasive potential of HMHA1 wildtype cancer cells in ROS- and HIF-dependent manners, but not of HMHA1 knockout cells. CONCLUSION: These results together provide insights into a potential molecular mechanism of the acquisition of invasiveness by hypoxic cancer cells after radiotherapy via the activation of the ROS/HIF/HMHA1 axis.

Downregulation of ABCC3 activates MAPK signaling through accumulation of deoxycholic acid in colorectal cancer cells.

ABCC3 (also known as MRP3) is an ATP binding cassette transporter for bile acids, whose expression is downregulated in colorectal cancer through the Wnt/ß-catenin signaling pathway. However, it remained unclear how downregulation of ABCC3 expression contributes to colorectal carcinogenesis. We explored the role of ABCC3 in the progression of colorectal cancer-in particular, focusing on the regulation of bile acid export. Gene expression analysis of colorectal adenoma isolated from familial adenomatous polyposis patients revealed that genes related to bile acid secretion including ABCC3 were downregulated as early as at the stage of adenoma formation. Knockdown or overexpression of ABCC3 increased or decreased intracellular concentration of deoxycholic acid, a secondary bile acid, respectively, in colorectal cancer cells. Forced expression of ABCC3 suppressed deoxycholic acid-induced activation of MAPK signaling. Finally, we found that nonsteroidal anti-inflammatory drugs increased ABCC3 expression in colorectal cancer cells, suggesting that ABCC3 could be one of the targets for therapeutic intervention of familial adenomatous polyposis. Our data thus suggest that downregulation of ABCC3 expression contributes to colorectal carcinogenesis through the regulation of intracellular accumulation of bile acids and activity of MAPK signaling.

Whole-Body and Whole-Organ 3D Imaging of Hypoxia Using an Activatable Covalent Fluorescent Probe Compatible with Tissue Clearing.

Elucidation of biological phenomena requires imaging of microenvironments in vivo. Although the seamless visualization of in vivo hypoxia from the level of whole-body to single-cell has great potential to discover unknown phenomena in biological and medical fields, no methodology for achieving it has been established thus far. Here, we report the whole-body and whole-organ imaging of hypoxia, an important microenvironment, at single-cell resolution using activatable covalent fluorescent probes compatible with tissue clearing. We initially focused on overcoming the incompatibility of fluorescent dyes and refractive index matching solutions (RIMSs), which has greatly hindered the development of fluorescent molecular probes in the field of tissue clearing. The fluorescent dyes compatible with RIMS were then incorporated into the development of activatable covalent fluorescent probes for hypoxia. We combined the probes with tissue clearing, achieving comprehensive single-cell-resolution imaging of hypoxia in a whole mouse body and whole organs.

A reductively convertible nickel phthalocyanine precursor as a biological thiol-responsive turn-on photoacoustic contrast agent.

A nickel phthalocyanine precursor bearing poly(ethylene glycol) as a turn-on contrast agent for photoacoustic imaging was prepared. The water-soluble polymeric chains were smoothly eliminated through thiol-mediated reductive aromatization in cancer cells, enabling the detection of endogenous biological thiols in vitro and in vivo.

DDX5 enhances HIF-1 activity by promoting the interaction of HIF-1α with HIF-1ß and recruiting the resulting heterodimer to its target gene loci.

BACKGROUND INFORMATION: Cancer cells acquire malignant characteristics and therapy resistance by employing the hypoxia-inducible factor 1 (HIF-1)-dependent adaptive response to hypoxic microenvironment in solid tumors. Since the underlying molecular mechanisms remain unclear, difficulties are associated with establishing effective therapeutic strategies. RESULTS: We herein identified DEAD-box helicase 5 (DDX5) as a novel activator of HIF-1 and found that it enhanced the heterodimer formation of HIF-1α and HIF-1ß and facilitated the recruitment of the resulting HIF-1 to its recognition sequence, hypoxia-response element (HRE), leading to the expression of a subset of cancer-related genes under hypoxia. CONCLUSIONS: This study reveals that the regulation of HIF-1 recruitment to HRE is an important regulatory step in the control of HIF-1 activity. SIGNIFICANCE: The present study provides novel insights for the development of strategies to inhibit the HIF-1-dependent expression of cancer-related genes.

ZBTB2 is Recruited to a Specific Subset of HIF-1 Target Loci to Facilitate Full Gene Expression Under Hypoxia.

The cellular response to hypoxia is mainly governed by a transcription factor, hypoxia-inducible factor 1 (HIF-1). Although upregulation of HIF-1 target genes has been hypothesized to require interaction of HIF-1 with other coactivators, much remains to be elucidated regarding the underlying mechanisms. Here, we demonstrate that zinc finger and BTB domain-containing protein 2 (ZBTB2) enhances the expression of certain HIF-1 target genes under hypoxia. ChIP-Seq analysis showed that there is a subset of HIF-1 target genes with overlapping HIF-1 and ZBTB2 peaks. Examination of a representative gene, EGFR antisense RNA 1 (EGFR-AS1), showed that HIF-1 binding to the consensus hypoxia-responsive element (HRE) sequence resulted in the recruitment of ZBTB2 to the gene locus and increased p300-mediated histone acetylation, leading to enhanced gene expression under hypoxia. In contrast, expression of HIF-1 target genes lacking ZBTB2 peaks, such as carbonic anhydrase 9 (CA9), was not upregulated by ZBTB2. These findings demonstrate that ZBTB2 is a novel factor that can be recruited to the vicinity of HREs on a subset of HIF-1 target gene loci, and is required for their full expression under hypoxia.

Infliximab Inhibits Colitis Associated Cancer in Model Mice by Downregulating Genes Associated with Mast Cells and Decreasing Their Accumulation.

Inflammatory bowel diseases (IBDs), such as Crohn's disease or ulcerative colitis, can be treated with anti TNF-alpha (TNF-α) antibodies (Abs), but they also put patients with IBDs at risk of cancer. We aimed to determine whether the anti TNF-α Ab induces colon cancer development in vitro and in vivo, and to identify the genes involved in colitis-associated cancer. We found that TNF-α (50 ng/mL) inhibited the proliferation, migration, and invasion of HCT8 and COLO205 colon cancer cell lines and that anti TNF-α Ab neutralized TNF-α inhibition in vitro. The effects of anti TNF-α Ab, infliximab (10 mg/kg) were investigated in mouse models of colitis-associated cancer induced by intraperitoneally injected azoxymethane (AOM: 10 mg/kg)/orally administered dextran sodium sulfate (DSS: 2.5%) (AOM/DSS) in vivo. Infliximab significantly attenuated the development of colon cancer in these mice. Microarray analyses and RT-qPCR revealed that mast cell protease 1, mast cell protease 2, and chymase 1 were up-regulated in cancer tissue of AOM/DSS mice; however, those mast cell related genes were downregulated in cancer tissue of AOM/DSS mice with infliximab. These results suggested that mast cells play a pivotal role in the development of cancer associated with colitis in AOM/DSS mice.

Prostate-specific antigen kinetics contributes to decision making for biopsy referral: the predictive implication for PSA retest in patients with elevated PSA levels.

Background: It is common to repeat prostate-specific antigen (PSA) measurements for men with PSA elevation before prostate biopsy. In this scenario, they may have considerable psychological distress in fear of the presence of cancer until retests. We assessed possible clinical factors causing transient PSA rise and explored the parameters predictive of subsequent PSA change. Methods: As interfering conditions, the history of ejaculation, bicycling, and any types of infections were assessed using the questionnaire. The pattern of PSA change was compared in association with the various clinical factors. Predictive significance of PSA kinetics such as coefficient of variation (CV) and PSA velocity (PSAV) for PSA values at retest was evaluated. Results: The rate of reversion to the normal range was 38.3% at retest. The rate of 12.8% of men showed a large increase by ≥20%, whereas 38.2% of men showed a large decline by ≥20% from the baseline. Men with younger age (≤60 years), small prostate (<20 cc), and prior history of ejaculation or infections showed significantly larger PSA decrease than their counterparts. Those with large CV or PSAV before the baseline more frequently showed PSA decrease below the age-specific cutoff or decline by ≥10% from the baseline at retest. These parameters associated with PSA kinetics had independent predictive values for relevant PSA change at retest. Conclusions: Ejaculation and any types of infections should be avoided before PSA tests. Men with large PSA fluctuation before the baseline are likely to show a significant PSA decrease at retest. This predictive information may help both physicians to determine whether to proceed to an immediate biopsy and patients to reduce their psychological burden.

Syndrome of inappropriate secretion of antidiuretic hormone after total proctocolectomy for ulcerative colitis.

We report a case of the syndrome of inappropriate secretion of antidiuretic hormone (SIADH) after total proctocolectomy followed with ileal pouch-anal anastomosis (TPC-IPAA) for ulcerative colitis (UC). The patient was a 46-year-old woman. She was diagnosed with UC of pancolitis in 2000. High grade dysplasia was detected in the transverse colon after a surveillance colonoscopy in 2021. She underwent laparoscopy-assisted TPC-IPAA. On the sixth postoperative day, she had a decreased level of consciousness that worsened on the following day. Her laboratory data showed a serum sodium level of 108 mEq/L and the plasma osmolality was 234 mOsm/kg. We did not find any other abnormalities in the laboratory examination that could cause hyponatremia. Computed tomography scan showed no central nervous system disturbances such as a pituitary tumor, antidiuretic hormone-producing tumors, or pulmonary diseases. The patient was diagnosed with Syndrome of inappropriate secretion of antidiuretic hormone (SIADH) caused by surgical invasion. We started to administer 3% sodium chloride slowly to improve the hyponatremia. Her serum sodium level became normal and stable. Although it is rare for SIADH to be caused by abdominal surgery, if hyponatremia is observed after surgery, the possibility of postoperative SIADH should be considered.

ZBTB2 links p53 deficiency to HIF-1-mediated hypoxia signaling to promote cancer aggressiveness.

Aberrant activation of the hypoxia-inducible transcription factor HIF-1 and dysfunction of the tumor suppressor p53 have been reported to induce malignant phenotypes and therapy resistance of cancers. However, their mechanistic and functional relationship remains largely unknown. Here, we reveal a mechanism by which p53 deficiency triggers the activation of HIF-1-dependent hypoxia signaling and identify zinc finger and BTB domain-containing protein 2 (ZBTB2) as an important mediator. ZBTB2 forms homodimers via its N-terminus region and increases the transactivation activity of HIF-1 only when functional p53 is absent. The ZBTB2 homodimer facilitates invasion, distant metastasis, and growth of p53-deficient, but not p53-proficient, cancers. The intratumoral expression levels of ZBTB2 are associated with poor prognosis in lung cancer patients. ZBTB2 N-terminus-mimetic polypeptides competitively inhibit ZBTB2 homodimerization and significantly suppress the ZBTB2-HIF-1 axis, leading to antitumor effects. Our data reveal an important link between aberrant activation of hypoxia signaling and loss of a tumor suppressor and provide a rationale for targeting a key mediator, ZBTB2, to suppress cancer aggressiveness.

Plasma progastrin-releasing peptide level shows different predictive profiles for treatment response by androgen receptor axis-targeted agents in patients with metastatic castration-resistant prostate cancer.

BACKGROUND: The neuroendocrine (NE) pathway cannot be ignored as a mechanism for castration-resistant prostate cancer (CRPC) progression. The neuromediator, gastrin-releasing peptide (GRP) may be involved in the aberrant activation of the normal androgen receptor (AR) and increased AR variants. This study focused on plasma levels of progastrin-releasing peptide (ProGRP) and examined the treatment outcomes with androgen receptor axis-targeted (ARAT) agents. METHODS: One hundred patients with metastatic CRPC were enrolled. Enzalutamide (ENZ) or abiraterone acetate/prednisone (AA/P) were administered to 50 patients each in a nonrandomized manner as a first-line or later choice. Plasma ProGRP levels were determined using a chemiluminescent enzyme immunoassay, and data were collected prospectively. The study endpoints were prostate-specific antigen (PSA) response and survival estimates. RESULTS: In the ENZ series, ProGRP levels correlated with the maximum PSA change from baseline (high ProGRP: -34.5% vs. low ProGRP: -85.7% p = .033). PSA progression-free survival (PFS), radiographic/symptomatic (r/s) PFS, and overall survival (OS) in patients with high ProGRP were significantly worse than those in patients with low ProGRP (median PSA-PFS: 3.3 vs. 10.0 months, p = .001, r/s PFS: 5.0 vs. 15.0 months, p < 0.001, and OS 17.5 vs. 49.0 months, p < .001, respectively). In addition, ProGRP showed an independent predictive value for all survival estimates in multivariate analyses. In the AA/P series, ProGRP levels did not correlate with the PSA change or predict PSA-PFS and r/s PFS, but they maintained a significant difference in OS (19.0 vs. 48.0 months, p = .003). CONCLUSIONS: Plasma ProGRP provides a consistent predictive value for OS in metastatic CRPC patients who underwent therapy with ARAT agents. Meanwhile, ProGRP showed different predictive profiles for PSA- and r/s PFS between ENZ and AA/P. These findings clinically suggest a mechanism for CRPC progression involving the NE pathway via the GRP. The underlying mechanism of different predictive profiles by the ARAT agent should be explored in future research.

A case of intestinal malrotation apparent after laparoscopically total proctocolectomy followed by ileal-pouch-anal anastomosis for ulcerative colitis.

Intestinal malrotation (IM) is an abnormality due to a failure of the normal midgut rotation and fixation. We report a case of 46-year-old man with ulcerative colitis whose IM was apparent after laparoscopically total proctocolectomy (TPC) followed by ileal-pouch-anal anastomosis (IPAA) and ileostomy. There was no abnormal anatomy except for mobile cecum/ascending colon during the initial operation. Intestinal obstruction occurred after ileostomy closure. The computed tomography scan showed the duodeno-jejunal transition was located in right abdomen, the superior mesenteric vein was located left of the superior mesenteric artery (SMA) and the obstruction point was the distal ileum near the pouch. We performed an ileo-ileo bypass across the ventral side of the SMA to relieve the intestinal obstruction. The patient would have incomplete IM preoperatively, which became apparent by TPC. In case of TPC for mobile colon, anatomy of small intestine should be checked before IPAA.

Correlation between antibiotic use and antibiotic resistance: A multicenter study using the Japan Surveillance for Infection Prevention and Healthcare Epidemiology (J-SIPHE) system in Hokkaido, Japan.

BACKGROUND: The Japan Surveillance for Infection Prevention and Healthcare Epidemiology (J-SIPHE) system aggregates information related to antimicrobial resistance (AMR) measures in participating medical institutions nationwide and is intended to be used for promotion of AMR measures in participating facilities and their communities. This multicenter study aimed to determine the usefulness of the J-SIPHE system for evaluating the correlation between antibiotic use and antibiotic resistance in Hokkaido, Japan. METHODS: Data on antibiotic use and detection rate of major resistant Gram-negative bacteria at 19 hospitals in 2020 were collected from the J-SIPHE system, and data correlations were analyzed using JMP Pro. RESULTS: The detection rate of carbapenem-resistant Pseudomonas aeruginosa was significantly positively correlated with carbapenem use (Spearman's ρ = 0.551; P = .015). There were significant positive correlations between the detection rate of fluoroquinolone-resistant Escherichia coli and the use of piperacillin/tazobactam, carbapenems, and quinolones [ρ = 0.518 (P = .023), ρ = 0.76 (P < .001), and ρ = 0.502 (P = .029), respectively]. CONCLUSIONS: This is the first multicenter study to investigate the correlation between antibiotic use and antibiotic resistance using the J-SIPHE system. The results suggest that using this system may be beneficial for promoting AMR measures.

Circulating tumor cells and cell-free tumor DNA analyses in urothelial cancer using the LiquidBiopsy platform.

Background: Emerging data suggested that liquid biopsy such as detection of circulating tumor cells (CTCs) and cell-free tumor DNA analysis augments the management of patients with urothelial cancer (UC). We presented our pilot experience of liquid biopsy using the Ion Torrent platform to detect CTCs and genomic alterations in UC. Materials and methods: Blood or urine samples from 16 patients were subjected to CTC and plasma/urine cell-free tumor DNA isolation for next generation sequencing (NGS) using the Ion S5 system to detect mutations among 50 oncogenes on the Ion AmpliSeq Cancer Hotspot Panel. Results: The Ion Torrent platform detected a higher number of CTCs than those in previous studies using the CellSearchTM system. Overall, mutations were detected in 13/16 (81.3%) patients with a median number of 18 (range 12-25). NGS isolated 17 hotspot mutations from 11 genes and 41 novel genomic alterations from 24 genes, some of which are supposed to be clinically actionable. Conclusions: The Ion Torrent platform efficiently detected CTCs compared with previous reports. NGS with the present system also allowed for detection of gene alterations which are likely to be therapeutic targets and provided an attractive tool to guide personalized therapy for patients with advanced UC.

Proteolysis of a histone acetyl reader, ATAD2, induces chemoresistance of cancer cells under severe hypoxia by inhibiting cell cycle progression in S phase.

Cancer cells acquire chemoresistance in hypoxic regions of solid tumors, which is suggested to be at least partly due to reduction of their proliferative activity. However, molecular mechanisms behind it have not been fully elucidated. Here, we revealed the importance of active proteolysis of a histone acetylation reader, ATPase family AAA domain containing 2 (ATAD2), under hypoxia. We found that inactivation of an O2/Fe2+/α-ketoglutarate-dependent dioxygenase triggered ATAD2 proteolysis by the proteasome system upon severe hypoxia in a hypoxia-inducible factors (HIFs)-independent manner. Consistently, ATAD2 expression levels were markedly lower in perinecrotic hypoxic regions in both xenografted and clinical tumor tissues. The ATAD2 proteolysis was accompanied by a decrease in the amount of acetylated histone H3 lysine 27 and inhibited cell cycle progression from the early to late S phase under severe hypoxia. The retardation of S phase progression induced chemoresistance, which was blocked by overexpression of ATAD2. Together, these results indicate that ATAD2 proteolysis upon severe hypoxia induces chemoresistance of cancer cells through heterochromatinization and the subsequent retardation of S phase progression; therefore, inhibition of ATAD2 proteolysis is expected to be a strategy to overcome chemoresistance of hypoxic tumor cells.

Epidemiological estimates and early detection of polycystic kidney disease by ultrasonographic assessment in Japan.

AIMS: To estimate the prevalence of autosomal dominant polycystic kidney disease (ADPKD) and provide the evaluation of new ultrasonographic criteria and clinical indicators to help its early detection. MATERIALS AND METHODS: A total of 30750 individuals for health check-up with abdominal ultrasonography (US) were included, in which 231 suspects of ADPKD based on the number of renal cysts were extracted. They were divided into 4 groups by the grade of suspicion (definitive, a strong suspect, a fair suspect and a weak suspect). Longitudinal data of US and renal function tests were compared between the groups.The estimated prevalence rate was 0.068% from the study subjects. The level of eGFR did not differ between the definitive and suspects, while the annual estimated glomerular filtration ratio (eGFR) decline was significantly larger in the former (p<0.001). The subjects with growing renal cysts showed a larger annual eGFR decline than those without growth (p=0.0324). The proposed cut-off set at the first quartile of the annualized eGFR change efficiently divided the subjects according to the presence of cyst growth (p= 0.027) and the grade of suspicion of ADPKD (p=0.028). CONCLUSION: The prevalence rate of ADPKD was higher than the corresponding rate previously reported in Japan (0.025%), suggesting that health check-ups may be an efficient opportunity to pick up undiagnosed ADPKD. The large annual eGFR decline and the presence of growing cysts may be feasible indicators to isolate ADPKD and should be introduced into US based screening to facilitate early detectionof ADPKD.

[A Case of Laparoscopic Abdominoperineal Resection and Perineal Reconstruction for Signet-Ring Cell Carcinoma with Skin Invasion Derived from the Anal Gland].

A 56-year-old man was referred to our hospital with an awareness of anal tumor. The tumor extended from the anal verge to the back of left testicle. Colonoscopy showed no tumor in the rectum and the anal canal. Biopsy showed mucus- producing adenocarcinoma(sig), and we diagnosed anal canal adenocarcinoma with immunostaining. Laparoscopic abdominoperineal rectal resection and perineal reconstruction with the V-Y fasciocutaneous flap closure technique. The patient had no major postoperative complications, and was discharged on 23rd postoperative day. Pathological examination revealed that the tumor was pT3N0M0, pStage ⅡB. The patient received adjuvant chemotherapy with CAPOX and has survived 12 months without recurrence. Immunostaining may be used to diagnose the signet-ring cell carcinoma without tumor of anal canal. In addition, reconstruction of the perineum for large anal tumors is useful.

[A Case of Robot-Assisted Abdominoperineal Resection with Prostatectomy for Locally Advanced Rectal Cancer].

The patient was referred to our hospital because of bloody stool and anorectal pain, and a colonoscopy revealed a tumor in the lower rectum. Although no distant metastasis was found, the tumor was suspected to have invaded the distal prostate. Neoadjuvant chemoradiotherapy(45 Gy/25 Fr with S-1)resulted in tumor shrinkage and symptomatic improvement, however, the primary tumor remained in close proximity to the prostate and urethra. Thus, we performed a robot-assisted abdominoperineal resection and Retzius-sparing prostatectomy in collaboration with the urology department. The surgical margins were negative and radical resection was achieved. Although minor vesicourethral anastomotic leakage was observed, it recovered conservatively. The patient has been alive 1 year postoperatively without recurrence. The patient initially had urinary incontinence, but it gradually improved. Although a total pelvic resection could have been considered, the robot-assisted surgery made it possible to preserve the urinary tract. The future application of robot-assisted surgery in extended surgery is expected.

SPINK1 as a plasma marker for tumor hypoxia and a therapeutic target for radiosensitization.

Hypoxia is associated with tumor radioresistance; therefore, a predictive marker for tumor hypoxia and a rational target to overcome it have been sought to realize personalized radiotherapy. Here, we show that serine protease inhibitor Kazal type I (SPINK1) meets these 2 criteria. SPINK1 expression was induced upon hypoxia (O2 < 0.1%) at the transcription initiation level in a HIF-dependent manner, causing an increase in secreted SPINK1 levels. SPINK1 proteins were detected both within and around hypoxic regions of xenografted and clinical tumor tissues, and their plasma levels increased in response to decreased oxygen supply to xenografts. Secreted SPINK1 proteins enhanced radioresistance of cancer cells even under normoxic conditions in EGFR-dependent and nuclear factor erythroid 2-related factor 2-dependent (Nrf2-dependent) manners and accelerated tumor growth after radiotherapy. An anti-SPINK1 neutralizing antibody exhibited a radiosensitizing effect. These results suggest that SPINK1 secreted from hypoxic cells protects the surrounding and relatively oxygenated cancer cells from radiation in a paracrine manner, justifying the use of SPINK1 as a target for radiosensitization and a plasma marker for predicting tumor hypoxia.