Interaction between TBP and Condensin Drives the Organization and Faithful Segregation of Mitotic Chromosomes.

Genome/chromosome organization is highly ordered and controls various nuclear events, although the molecular mechanisms underlying the functional organization remain largely unknown. Here, we show that the TATA box-binding protein (TBP) interacts with the Cnd2 kleisin subunit of condensin to mediate interphase and mitotic chromosomal organization in fission yeast. TBP recruits condensin onto RNA polymerase III-transcribed (Pol III) genes and highly transcribed Pol II genes; condensin in turn associates these genes with centromeres. Inhibition of the Cnd2-TBP interaction disrupts condensin localization across the genome and the proper assembly of mitotic chromosomes, leading to severe defects in chromosome segregation and eventually causing cellular lethality. We propose that the Cnd2-TBP interaction coordinates transcription with chromosomal architecture by linking dispersed gene loci with centromeres. This chromosome arrangement can contribute to the efficient transmission of physical force at the kinetochore to chromosomal arms, thereby supporting the fidelity of chromosome segregation.

Simple Detection and Culture of Circulating Tumor Cells from Colorectal Cancer Patients Using Poly(2-Methoxyethyl Acrylate)-Coated Plates.

Here we aimed to establish a simple detection method for detecting circulating tumor cells (CTCs) in the blood sample of colorectal cancer (CRC) patients using poly(2-methoxyethyl acrylate) (PMEA)-coated plates. Adhesion test and spike test using CRC cell lines assured efficacy of PMEA coating. A total of 41 patients with pathological stage II-IV CRC were enrolled between January 2018 and September 2022. Blood samples were concentrated by centrifugation by the OncoQuick tube, and then incubated overnight on PMEA-coated chamber slides. The next day, cell culture and immunocytochemistry with anti-EpCAM antibody were performed. Adhesion tests revealed good attachment of CRCs to PMEA-coated plates. Spike tests indicated that ~75% of CRCs from a 10-mL blood sample were recovered on the slides. By cytological examination, CTCs were identified in 18/41 CRC cases (43.9%). In cell cultures, spheroid-like structures or tumor-cell clusters were found in 18/33 tested cases (54.5%). Overall, CTCs and/or growing circulating tumor cells were found in 23/41 CRC cases (56.0%). History of chemotherapy or radiation was significantly negatively correlated with CTC detection (p = 0.02). In summary, we successfully captured CTCs from CRC patients using the unique biomaterial PMEA. Cultured tumor cells will provide important and timely information regarding the molecular basis of CTCs.

A stem cell marker KLF5 regulates CCAT1 via three-dimensional genome structure in colorectal cancer cells.

BACKGROUND: KLF5 plays a crucial role in stem cells of colorectum in cooperation with Lgr5 gene. In this study, we aimed to explicate a regulatory mechanism of the KLF5 gene product from a view of three-dimensional genome structure in colorectal cancer (CRC). METHODS: In vitro engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP)-seq method was used to identify the regions that bind to the KLF5 promoter. RESULTS: We revealed that the KLF5 promoter region interacted with the KLF5 enhancer region as well as the transcription start site (TSS) region of the Colon Cancer Associated Transcript 1 (CCAT1) gene. Notably, the heterodeletion mutants of KLF5 enhancer impaired the cancer stem-like properties of CRC cells. The KLF5 protein participated in the core-regulatory circuitry together with co-factors (BRD4, MED1, and RAD21), which constructs the three-dimensional genome structures consisting of KLF5 promoter, enhancer and CCAT1 TSS region. In vitro analysis indicated that KLF5 regulated CCAT1 expression and we found that CCAT1 expression was highly correlated with KLF5 expression in CRC clinical samples. CONCLUSIONS: Our data propose the mechanistic insight that the KLF5 protein constructs the core-regulatory circuitry with co-factors in the three-dimensional genome structure and coordinately regulates KLF5 and CCAT1 expression in CRC.

miR-4711-5p regulates cancer stemness and cell cycle progression via KLF5, MDM2 and TFDP1 in colon cancer cells.

BACKGROUND: It is important to establish cancer stem cell (CSC)-targeted therapies to eradicate cancer. As it is a CSC marker, we focused on Kruppel-like factor 5 (KLF5) in this study. METHODS: We searched for candidate microRNAs (miRNAs) that inhibited KLF5 expression by in silico analyses and screened them in colon cancer cell lines. RESULTS: We identified one promising miRNA, miR-4711-5p, that downregulated KLF5 expression by direct binding. This miRNA suppressed cell proliferation, migration and invasion ability, as well as stemness, including decreased stem cell marker expression, reactive oxygen species activity and sphere formation ability. MiR-4711-5p inhibited the growth of DLD-1 xenografts in nude mice with no adverse effects. We found that miR-4711-5p provoked G1 arrest, which could be attributed to direct binding of miR-4711-5p to TFDP1 (a heterodimeric partner of the E2F family). Our findings also suggested that direct binding of miR-4711-5p to MDM2 could upregulate wild-type p53, leading to strong induction of apoptosis. Finally, we found that miR-4711-5p had a potent tumour-suppressive effect compared with a putative anti-oncomiR, miR-34a, in tumour cell cultures derived from five patients with colorectal cancer. CONCLUSIONS: Our data suggest that miR-4711-5p could be a promising target for CSC therapy.

[Dendritic Cell Therapy with Neoantigens and Oncoantigens].

We treated 3,164 patients with advanced cancer with dendritic cell therapy between July 2005 and March 2020. The effective rate in patients treated with dendritic cell therapy more than 3 times was 19.0%. Among them, we treated 133 cancer patients with a combination of immune checkpoint inhibitors and dendritic cell therapy between June 2015 and March 2020. The effective rate in these patients was 54.1%. We treated 98 cancer patients with dendritic cell therapy with neoantigens between March 2018 and March 2020. The effective rate in these patients treated with neoantigens was 38.7%. The effective rate in patients treated without neoantigens was 18.3%. Dendritic cell therapy with neoantigens enhanced the effective rate. The effective rate of dendritic cell therapy with both immune checkpoint inhibitors and neoantigens was 60.7%.

E-cadherin-Fc chimera protein matrix enhances cancer stem-like properties and induces mesenchymal features in colon cancer cells.

Cancer stem cells (CSC) are a subpopulation of tumor cells with properties of high tumorigenicity and drug resistance, which lead to recurrence and poor prognosis. Although a better understanding of CSC is essential for developing cancer therapies, scarcity of the CSC population has hindered such analyses. The aim of the present study was to elucidate whether the E-cadherin-Fc chimera protein (E-cad-Fc) enhances cancer stem-like properties because studies show that soluble E-cadherin stimulates human epithelial growth factor receptor (EGFR) and downstream signaling pathways that are reported to play a crucial role in CSC. For this purpose, we used ornithine decarboxylase (ODC)-degron-transduced (Degron(+)) KM12SM cells as a CSC model that retains relatively low CSC properties. Compared to cultures without E-cad-Fc treatment, we found that E-cad-Fc treatment further suppressed proteasome activity and largely enhanced cancer stem-like properties of ODC-degron-transduced KM12SM cells. These results include increased expression of stem cell markers Lgr5, Bmi-1, SOX9, CD44, and CD44v9, aldehyde dehydrogenase (ALDH), and enhancement of robust spheroid formation, and chemoresistance to 5-fluorouracil (5-FU) and oxaliplatin (L-OHP). These effects could be attributed to activation of the EGFR pathway as identified by extensive phosphorylation of EGFR, ERK, PI3K, AKT, and mTOR. In SW480 cells, E-cad-Fc matrix induced some CSC markers such as CD44v9 and ALDH. We also found that E-cad-Fc matrix showed high efficiency of inducing mesenchymal changes in colon cancer cells. Our data suggest that the E-cad-Fc matrix may enhance CSC properties such as enhancement of chemoresistance and sphere formation.

[Combination of Immunotherapy and Hyperthermia].

We treated 123cancer patients with a combination of immune checkpoint inhibitor and dendritic cell therapy between June 2015 and April 2019. The effective rate of cases administered for B3times was 51.5%. Hyperthermia was found to enhance the effects of radiotherapy, chemotherapy, and immunotherapy. In addition, hyperthermia enhanced the effects of combined immunotherapy. In clinical cases and animal models, immunohistochemical staining showed that the expression of PD-L1 and MHC classⅠ and invasion of CD8 cells were increased after hyperthermia.

[Effects of Immune Checkpoint Inhibitor Combined with Dendritic Cell Therapy and Hyperthermia].

In this study, 97cancer patients were treated with combined immune checkpoint inhibitor therapy and dendritic cell thera- py between June 2015 and April 2018. We administered nivolumab with 2-3mg/kg bw every 2-3weeks. The rate of progress in cases where nivolumab was administered more than 3 times was 55.2%. Dendritic cell therapy enhanced the immune checkpoint inhibitor therapy. In cases where the effect of combined immune checkpoint inhibitor therapy and dendritic cell therapy was restricted, hyperthermia and radiation therapy are useful for the recovery of combined therapy.

[Enhancing Antitumor Effects Using Hyperthermia(Including Immune Checkpoint Inhibitor)].

Hyperthermia enhances the efficacies of radiotherapy, chemotherapy, immunotherapy, and molecular targeted therapy. In this study, we investigated whether hyperthermia enhanced the efficacy of immune check point inhibitor therapy. The LLC tumor inoculated in mouse was heated and immunostained, which showed increase in PD-L1 staining post-heating. PD-L1 and HLA class I staining increased after heating samples derived from a case of analinvasion of rectalcancer associated with Lynch syndrome.

SUV420H2 suppresses breast cancer cell invasion through down regulation of the SH2 domain-containing focal adhesion protein tensin-3.

The genome-wide loss of histone H4 lysine 20 tri-methylation (H4K20me3) is observed in multiple types of cancer, including breast tumors. Since H4K20me3 is preferentially targeted to repetitive elements in the pericentromeric and telomeric heterochromatin and plays a role in chromatin integrity, the pathological effects of disrupted H4K20me3 in tumors have been attributed to genomic instability. However, in this report, we show that loss of H4K20me3 modulates gene expression profiles, leading to increased cell invasion. Reduced H4K20me3 levels in tumor cells are often accompanied by a decrease in the expression of the H4K20-specific methyltransferase, SUV420H2. Exogenous delivery of SUV420H2 into MDA-MB-231 human breast cancer cells induced selective and specific changes in the expression of cancer-related genes. One of the most downregulated genes in response to SUV420H2 expression was the Src substrate, tensin-3, a focal adhesion protein that contributes to cancer cell migration. Depletion of tensin-3 suppressed breast cancer cell invasiveness. Furthermore, silencing of tensin-3 was associated with enrichment of H4K20me3 immediately upstream of the tensin-3 transcription start site, suggesting that the loss of H4K20me3 in tumor cells induced the expression of cancer-promoting genes. These findings connect the loss of H4K20me3 with tumor progression, through the transcriptional activation of cancer-promoting genes.

Multicenter large-scale study of prognostic impact of HER2 expression in patients with resectable gastric cancer.

BACKGROUND: Although some small-scale studies have suggested that human epidermal growth factor receptor 2 (HER2)-positive status in gastric cancer is associated with poor outcomes, the prognostic value of HER2 is still controversial. Since intratumoral HER2 heterogeneity is also an important issue, a multicenter large-scale study was conducted to evaluate the prognostic impacts of HER2 expression and intratumoral heterogeneity in gastric cancer. METHODS: This study included 1,148 gastric cancer patients who underwent gastrectomy in 11 institutions. HER2 expression was centrally evaluated with immunohistochemistry and fluorescence in situ hybridization, and intratumoral HER2 heterogeneity was evaluated for HER2-positive tumors. Overall survival was compared between HER2-positive and HER2-negative patients and between the homogeneous and heterogeneous groups. RESULTS: The HER2-positive rate was 15.7 %, and HER2 expression was significantly associated with histological type. HER2 expression scores obtained by immunohistochemistry showed a distinct influence on survival, and HER2-positive patients showed much poorer survival than HER2-negative patients [hazard ratio (HR) 1.59, 95 % confidence interval (CI) 1.24-2.02; P < 0.001). The subgroup analysis by pathological tumor stage showed a similar trend of poor survival in HER2-positive patients. Both intestinal type and diffuse type showed significant poor survival in HER2-positive patients. Cox multivariate analysis revealed that HER2 expression was an independent prognostic factor (HR 1.96, 95 % CI 1.51-2.55; P < 0.001). HER2 heterogeneity was observed in 75.4 % of HER2-positive cases, but the prognosis in the heterogeneous group was similar to that in the homogeneous group. CONCLUSIONS: Our study demonstrated that HER2 overexpression is an independent prognostic factor in patients with any stage of resectable gastric cancer. Intratumoral HER2 heterogeneity did not affect prognosis.

Loss of histone H4K20 trimethylation predicts poor prognosis in breast cancer and is associated with invasive activity.

INTRODUCTION: Loss of histone H4 lysine 20 trimethylation (H4K20me3) is associated with multiple cancers, but its role in breast tumors is unclear. In addition, the pathological effects of global reduction in H4K20me3 remain mostly unknown. Therefore, a major goal of this study was to elucidate the global H4K20me3 level in breast cancer tissue and investigate its pathological functions. METHODS: Levels of H4K20me3 and an associated histone modification, H3 lysine 9 trimethylation (H3K9me3), were evaluated by immunohistochemistry in a series of breast cancer tissues. Univariate and multivariate clinicopathological and survival analyses were performed. We also examined the effect of overexpression or knockdown of the histone H4K20 methyltransferases, SUV420H1 and SUV420H2, on cancer-cell invasion activity in vitro. RESULTS: H4K20me3, but not H3K9me3, was clearly reduced in breast cancer tissue. A reduced level of H4K20me3 was correlated with several aspects of clinicopathological status, including luminal subtypes, but not with HER2 expression. Multivariate analysis showed that reduced levels of H4K20me3 independently associated with lower disease-free survival. Moreover, ectopic expression of SUV420H1 and SUV420H2 in breast cancer cells suppressed cell invasiveness, whereas knockdown of SUV420H2 activated normal mammary epithelial-cell invasion in vitro. CONCLUSIONS: H4K20me3 was reduced in cancerous regions of breast-tumor tissue, as in other types of tumor. Reduced H4K20me3 level can be used as an independent marker of poor prognosis in breast cancer patients. Most importantly, this study suggests that a reduced level of H4K20me3 increases the invasiveness of breast cancer cells in a HER2-independent manner.

Prognostic impact of major receptor tyrosine kinase expression in gastric cancer.

BACKGROUND: Various kinds of molecular targeted drugs to inhibit receptor tyrosine kinases (RTKs) have been recently developed. The relationship between the expression status of major RTKs and prognosis in gastric cancer remains unclear. We conducted a multicenter study to evaluate the prognostic impact of the expression of epidermal growth factor receptor (EGFR), c-Met, platelet-derived growth factor receptor (PDGFR), and c-Kit in gastric cancer. METHODS: This study included 153 gastric cancer patients who underwent gastrectomy at 9 institutions between 2000 and 2006. Expression status of EGFR, c-Met, PDGFR, and c-Kit were evaluated with immunohistochemistry (IHC) centrally. Overall survival based on RTK expression status was statistically compared. Cox multivariate analysis was conducted to adjust for potentially confounding factors. RESULTS: The positive rates for EGFR, c-Met, PDGFR, and c-Kit were 14.4, 24.8, 41.2, and 11.1 %, respectively. Significant interactions with expression status were observed for pathological N stage with EGFR; HER2-status with c-Met; tumor location, histology, and pathological N stage with PDGFR; and no examined variables with c-Kit. Concomitant HER2 positivity was observed for 0.7 % of tumors positive for EGFR, 3.9 % for c-Met, 4.6 % for PDGFR, and 1.3 % for c-Kit. There were some differences in overall survival between patients with or without RTK expression, but only c-Kit expression showed a significant survival difference in Cox multivariate analysis (P = 0.046). CONCLUSIONS: Our multicenter study indicated that IHC expression of 4 RTKs had some prognostic impact and that c-Kit-positive status may be a significant indicator of good prognosis in gastric cancer patients.

Cancer-associated upregulation of histone H3 lysine 9 trimethylation promotes cell motility in vitro and drives tumor formation in vivo.

Global histone modification patterns correlate with tumor phenotypes and prognostic factors in multiple tumor types. Recent studies suggest that aberrant histone modifications play an important role in cancer. However, the effects of global epigenetic rearrangements on cell functions remain poorly understood. In this study, we show that the histone H3 lysine 9 (H3K9) methyltransferase SUV39H1 is clearly involved in regulating cell migration in vitro. Overexpression of wild-type SUV39H1, but not enzymatically inactive SUV39H1, activated migration in breast and colorectal cancer cells. Inversely, migration was reduced by knockdown of SUV39H1 or chemical inhibition by chaetocin. In addition, H3K9 trimethylation (H3K9me3) was specifically increased in invasive regions of colorectal cancer tissues. Moreover, the presence of H3K9me3 positively correlated with lymph node metastasis in colorectal cancer patients. Furthermore, overexpression of SUV39H1 drove tumorigenesis in mouse, resulting in a considerable decrease in survival rate. These data indicate that H3K9 trimethylation plays an important role in human colorectal cancer progression, possibly by promoting collective cell invasion.

Histone methyltransferase SUV39H1 regulates the Golgi complex via the nuclear envelope-spanning LINC complex.

Cell motility is related to the higher-order structure of chromatin. Stimuli that induce cell migration change chromatin organization; such stimuli include elevated histone H3 lysine 9 trimethylation (H3K9me3). We previously showed that depletion of histone H3 lysine 9 methyltransferase, SUV39H1, suppresses directional cell migration. However, the molecular mechanism underlying this association between chromatin and cell migration remains elusive. The Golgi apparatus is a cell organelle essential for cell motility. In this study, we show that loss of H3K9 methyltransferase SUV39H1 but not SETDB1 or SETDB2 causes dispersion of the Golgi apparatus throughout the cytoplasm. The Golgi dispersion triggered by SUV39H1 depletion is independent of transcription, centrosomes, and microtubule organization, but is suppressed by depletion of any of the following three proteins: LINC complex components SUN2, nesprin-2, or microtubule plus-end-directed kinesin-like protein KIF20A. In addition, SUN2 is closely localized to H3K9me3, and SUV39H1 affects the mobility of SUN2 in the nuclear envelope. Further, inhibition of cell motility caused by SUV39H1 depletion is restored by suppression of SUN2, nesprin-2, or KIF20A. In summary, these results show the functional association between chromatin organization and cell motility via the Golgi organization regulated by the LINC complex.

KRT13 is upregulated in pancreatic cancer stem-like cells and associated with radioresistance.

Pancreatic cancer is one of the most aggressive cancers and the seventh leading cause of cancer-associated death in the world. Radiation is performed as an adjuvant therapy as well as anti-cancer drugs. Because cancer stem-like cells (CSCs) are considered to be radioresistant and cause recurrence and metastasis, understanding their properties is required for the development of novel therapeutic strategies. To investigate the CSC properties of pancreatic cancer cells, we used a pancreatic CSC model, degron (++) cells, which have low proteasome activity. Degron (++) cells displayed radioresistance in comparison with control cells. Using Ribonucleic acid (RNA) sequencing, we successfully identified KRT13 as a candidate gene responsible for radioresistance. Knockdown of KRT13 sensitized the degron (++) cells to radiation. Furthermore, a database search revealed that KRT13 is upregulated in pancreatic cancer cell lines and that high expression of KRT13 is associated with poorer prognosis. These results indicate that a combination therapy of KRT13 knockdown and radiation could hold therapeutic promise in pancreatic cancer.

The Relationship between LRP6 and Wnt/ß-Catenin Pathway in Colorectal and Esophageal Cancer.

High expression of low-density lipoprotein receptor-related protein 6 (LRP6), a key component of the Wnt/ß-catenin signaling pathway, is reported to be associated with malignant potential in some solid tumors including breast cancer and hepatocellular carcinoma. Few reports, however, have examined its function and clinical significance in colorectal cancers (CRC) demonstrating constitutive activation of Wnt signaling. Here, we compared the expression level and function of LRP6 in CRC with that of esophageal squamous cell carcinoma (ESCC) bearing few Wnt/ß-catenin pathway mutations. On immunohistochemical staining, high LRP6 expression was noted in three of 68 cases (4.4%), and high ß-catenin in 38 of 67 cases (56.7%) of CRC. High LRP6 expression was found in 21 of 82 cases (25.6%), and high ß-catenin expression in 29 of 73 cases (39.7%) of ESCC. In our in vitro studies, LRP6 knockdown hardly changed Wnt signaling activity in CRC cell lines with mutations in Wnt signaling downstream genes. In contrast, in ESCC cell lines without Wnt signaling-related mutations, LRP6 knockdown significantly decreased Wnt signaling activity. LRP6 function may depend on constitutive activation of Wnt signaling.

A quantitative evaluation method utilizing the homology concept to assess the state of chromatin within the nucleus of lung cancer.

Homology is a mathematical tool to quantify "the contact degree", which can be expressed in terms of Betti numbers. The Betti numbers used in this study consisted of two numbers, b0 (a zero-dimensional Betti number) and b1 (a one-dimensional Betti number). We developed a chromatin homology profile (CHP) method to quantify the chromatin contact degree based on this mathematical tool. Using the CHP method we analyzed the number of holes (surrounded areas = b1 value) formed by the chromatin contact and calculated the maximum value of b1 (b1MAX), the value of b1 exceeding 5 for the first time or Homology Value (HV), and the chromatin density (b1MAX/ns2). We attempted to detect differences in chromatin patterns and differentiate histological types of lung cancer from respiratory cytology using these three features. The HV of cancer cells was significantly lower than that of non-cancerous cells. Furthermore, b1MAX and b1MAX/ns2 showed significant differences between small cell and non-small cell carcinomas and between adenocarcinomas and squamous cell carcinomas, respectively. We quantitatively analyzed the chromatin patterns using homology and showed that the CHP method may be a useful tool for differentiating histological types of lung cancer in respiratory cytology.

Long noncoding RNA 01534 maintains cancer stemness by downregulating endoplasmic reticulum stress response in colorectal cancer.

Background: Studies have shown that cancer stemness and the endoplasmic reticulum (ER) stress response are inversely regulated in colorectal cancer (CRC), but the mechanism has not been fully clarified. Long noncoding RNAs (lncRNAs) play key roles in cancer progression and metastasis. In this study we investigated lncRNA 01534 (LINC01534) as a possible modulator between cancer stemness and ER stress response. Methods: In vitro experiments using CRC cell lines were performed to explore a possible role of LINC01534. The expression of LINC01534 in clinical CRC samples was assessed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and in situ hybridization. Results: Silencing LINC01534 led to suppression of cell proliferation, invasiveness, and cell cycle progression at the G2-M phase, and promoted apoptosis. Moreover, we found that silencing LINC01534 suppressed cancer stemness, while it activated the ER stress response, especially through the PERK/eIF2α signaling pathway. In situ hybridization revealed LINC01534 was expressed in tumor cells and upregulated in CRC tissues compared with normal epithelium. A survival survey indicated that high LINC01534 expression was significantly associated with shorter overall survival in 187 CRC patients. Conclusion: This is the first report on LINC01534 in human cancer. Our findings suggest that LINC01534 may be an important modulator of the maintenance of cancer stemness and suppression of the ER stress response, and that it could be a novel prognostic factor in CRC.

Single-Cell Analysis of Circulating Tumor Cells from Patients with Colorectal Cancer Captured with a Dielectrophoresis-Based Micropore System.

This study aimed to analyze circulating tumor cells (CTCs) from patients with colorectal cancer (CRC). We designed a dielectrophoresis-based micropore system and tested its cell capture with HT29 colon cancer cells. Then, blood samples were drawn from 24 patients with stages II-IV CRC. Mononuclear cells were isolated and loaded into the micropore system. Single cells were positioned into small pores with dielectrophoresis. After labeling the cells with the appropriate antibodies, tumor-like cells were collected with an automated micromanipulator. We collected 43 CTCs from 15 out of 24 patient samples. The presence of CTC was significantly associated with ling metastasis. We performed whole genome amplification, followed by PCR and Sanger sequencing, to examine the point mutations in the KRAS, BRAF, and PIK3CA genes. This mutation analysis was successfully performed in 35 cells. Among the 14 cytokeratin (CK)-positive cells, we found PIK3CA mutations in three cells (21%) from two patients. Among the 21 CK-negative cells, we found a KRAS mutation in one cell (5%) from one patient and a PIK3CA mutation in one cell (5%) from one patient. It is noteworthy that these mutations were not detected in the corresponding primary tumors. In conclusion, dielectrophoresis-based capture in a micropore system was useful for detecting both CK-positive and CK-negative CTCs. This simple method could be applied to various tumor types.