Cancer Stem Cells in Head and Neck Cutaneous Squamous Cell Carcinoma Express Cathepsins.

Cancer stem cell (CSC) subpopulations within moderately differentiated head and neck cutaneous squamous cell carcinoma (MDHNcSCC) express the components of the renin-angiotensin system (RAS). This study investigated the expression of cathepsins B, D, and G, which constitute bypass loops of the RAS, by CSCs in MDHNcSCC. METHODS: Immunohistochemical staining was performed on MDHNcSCC tissue samples from 15 patients to determine the expression of cathepsins B, D, and G. Co-localization of these cathepsins with the embryonic stem cell markers Octamer-binding transcription factor 4 (OCT4) and c-MYC was investigated with immunofluorescence staining. Reverse transcription quantitative polymerase chain reaction was performed on 5 MDHNcSCC tissue samples to investigate transcript expression of cathepsins B, D and G. Western blotting and enzymatic activity assays were performed on 5 MDHNcSCC tissue samples and 6 MDHNcSCC-derived primary cell lines to confirm protein expression, transcript expression, and functional activity of these cathepsins, respectively. RESULTS: Immunohistochemical staining demonstrated the expression of cathepsins B, D, and G in all MDHNcSCC tissue samples. Immunofluorescence staining showed localization of cathepsins B and D to the c-MYC+ CSC subpopulations and the OCT4+ CSC subpopulations within the tumor nests and the peritumoral stroma. Cathepsin G was expressed on the tryptase+/c-MYC+ cells within the peritumoral stroma. Reverse transcription quantitative polymerase chain reaction demonstrated transcript expression of cathepsins B, D and G in the MDHNcSCC tissue samples. Western blotting and enzymatic activity assays confirmed protein expression and functional activity of cathepsins B and D in the MDHNcSCC tissue samples and MDHNcSCC-derived primary cell lines, respectively. CONCLUSIONS: Cathepsins B, D, and G are expressed in MDHNcSCC with functionally active cathepsins B and D localizing to the CSC subpopulations, and cathepsin G is expressed by mast cells, suggesting the potential use of cathepsin inhibitors in addition to RAS blockade to target CSCs in MDHNcSCC.

Proliferating Infantile Hemangioma Tissues and Primary Cell Lines Express Markers Associated with Endothelial-to-Mesenchymal Transition.

BACKGROUND: We have previously shown that the endothelium of the microvessels of infantile hemangioma (IH) exhibits a hemogenic endothelium phenotype and proposed its potential to give rise to mesenchymal stem cells, similar to the development of hematopoietic cells. This endothelial-to-mesenchymal transition (Endo-MT) process involves the acquisition of a migratory phenotype by the endothelial cells, similar to epithelial-to-mesenchymal transition that occurs during neural crest development. We hypothesized that proliferating IH expresses Endo-MT-associated proteins and investigated their expression at the mRNA, protein, and functional levels. METHODS: Immunohistochemical staining of paraffin-embedded sections of proliferating IH samples from 10 patients was undertaken to investigate the expression of the Endo-MT proteins Twist1, Twist2, Snail1, and Slug. Transcriptional analysis was performed for the same markers on proliferating IH tissues and CD34+ and CD34- cells from proliferating IH-derived primary cell lines. Adipogenic and osteogenic differentiation plasticity was determined on the CD34-sorted fractions. RESULTS: The endothelium of the microvessels and the cells within the interstitium of proliferating IH tissues expressed Twist1, Twist2, and Slug proteins. Twist1 was also expressed on the pericyte layer of the microvessels, whereas Snail1 was not expressed. Both CD34+ and CD34- populations from the IH-derived primary cell lines underwent adipogenic and osteogenic differentiation. CONCLUSIONS: The expression of Endo-MT-associated proteins Twist1, Twist2, and Slug by both the endothelium of the microvessels and cells within the interstitium, and Twist1 on the pericyte layer of the microvessels of proliferating IH, suggest the presence of a process similar to Endo-MT. This may enable a tightly controlled primitive endothelium of proliferating IH to acquire a migratory mesenchymal phenotype with the ability to migrate away, providing a plausible explanation for the development of a fibrofatty residuum observed during involution of IH.

Expression of Cathepsins B, D, and G by the Embryonic Stem Cell-Like Population within Human Keloid Tissues and Keloid-Derived Primary Cell Lines.

BACKGROUND: The authors have previously shown that an embryonic stem cell-like population within keloid-associated lymphoid tissues in keloid lesions expresses components of the renin-angiotensin system that may be dysregulated. The authors hypothesized that cathepsins B, D, and G are present within the embryonic stem cell-like population in keloid lesions and contribute to bypass loops of the renin-angiotensin system. METHODS: 3,3'-Diaminobenzidine immunohistochemical staining for cathepsins B, D, and G was performed on formalin-fixed paraffin-embedded sections in keloid tissue samples of 11 patients. Immunofluorescence immunohistochemical staining was performed on three of these keloid tissue samples, by co-staining with CD34, tryptase, and OCT4. Western blotting, reverse transcription quantitative polymerase chain reaction, and enzyme activity assays were performed on five keloid tissue samples and four keloid-derived primary cell lines to investigate protein and mRNA expression, and functional activity, respectively. RESULTS: 3,3'-Diaminobenzidine immunohistochemical staining demonstrated expression of cathepsins B, D, and G in all 15 keloid tissue samples. Immunofluorescence immunohistochemical staining showed localization of cathepsins B and D to the endothelium of microvessels within the keloid-associated lymphoid tissues and localization of cathepsin G to the tryptase-positive perivascular cells. Western blotting confirmed semiquantitative levels of cathepsins B and D in keloid tissue samples and keloid-derived primary cell lines. Reverse transcription quantitative polymerase chain reaction showed quantitative transcriptional activation of cathepsins B and D in keloid tissue samples and keloid-derived primary cell lines and cathepsin G in keloid tissue samples. Enzyme activity assays demonstrated functional activity of cathepsins B and D. CONCLUSION: Cathepsins B, D, and G are expressed by the embryonic stem cell-like population within the keloid-associated lymphoid tissues of keloid lesions and may act to bypass the renin-angiotensin system, suggesting a potential therapeutic target using renin-angiotensin system modulators and cathepsin inhibitors.

Cancer stem cell subpopulations in primary colon adenocarcinoma.

AIMS: The cancer stem cell concept proposes that tumor growth and recurrence is driven by a small population of cancer stem cells (CSCs). In this study we investigated the expression of induced-pluripotent stem cell (iPSC) markers and their localization in primary low-grade adenocarcinoma (LGCA) and high-grade adenocarcinoma (HGCA) and their patient-matched normal colon samples. MATERIALS AND METHODS: Transcription and translation of iPSC markers OCT4, SOX2, NANOG, KLF4 and c-MYC were investigated using immunohistochemical (IHC) staining, RT-qPCR and in-situ hybridization (ISH). RESULTS: All five iPSC markers were detected at the transcriptional and translational levels. Protein abundance was found to be correlated with tumor grade. Based on their protein expression within the tumors, two sub-populations of cells were identified: a NANOG+/OCT4- epithelial subpopulation and an OCT4+/NANOG- stromal subpopulation. All cases were accurately graded based on four pieces of iPSC marker-related data. CONCLUSIONS: This study suggests the presence of two putative sub-populations of CSCs: a NANOG+/OCT4- epithelial subpopulation and an OCT4+/NANOG- stromal subpopulation. Normal colon, LGCA and HGCA could be accurately distinguished from one another using iPSC marker expression. Once validated, novel combinations of iPSC markers may provide diagnostic and prognostic value to help guide patient management.

Expression of Components of the Renin-Angiotensin System by the Putative Stem Cell Population Within WHO Grade I Meningioma.

Aim: We have recently demonstrated a putative stem cell population within WHO grade I meningioma (MG) that expressed embryonic stem cell (ESC) markers OCT4, NANOG, SOX2, KLF4 and c-MYC, localized to the endothelial and pericyte layers of the microvessels. There is increasing recognition that the renin-angiotensin system (RAS) plays a critical role in stem cell biology and tumorigenesis. This study investigated the expression of components of the RAS: pro-renin receptor (PRR), angiotensin converting enzyme (ACE), angiotensin II receptor 1 (ATIIR1), and angiotensin II receptor 2 (ATIIR2) on the putative stem cell population on the microvessels of WHO grade I MG. Methods: 3,3-Diaminobenzidine (DAB) immunohistochemical (IHC) staining was performed on WHO grade I MG tissue samples from 11 patients for PRR, ACE, ATIIR1, and ATIIR2. Two of the MG samples subjected to DAB IHC staining underwent immunofluorescence (IF) IHC staining to investigate co-expression of each of these components of the RAS in using combinations of CD34 and ESC marker SOX2 or OCT4. NanoString mRNA expression analysis and Western blotting (WB), were performed on six snap-frozen MG tissue samples to confirm mRNA and protein expression of these proteins, respectively. Results: DAB IHC staining demonstrated expression of PRR, ACE, ATIIR1, and ATIIR2 within all 11 MG tissue samples. WB and NanoString mRNA analyses, confirmed protein and mRNA expression of these proteins, respectively. IF IHC staining showed PRR, ATIIR1 and ATIIR2 were localized to the OCT4+ and SOX2+ endothelium and the pericyte layer of MG while ACE was localized to the OCT4+ endothelium of the microvesels. Conclusion: The novel finding of the expression of PRR, ACE, ATIIR1, and ATIIR2 on the putative stem cell population on the microvessels of WHO grade I MG, suggests that these stem cells may be a potential therapeutic target by manipulation of the RAS.

Expression of (pro)renin receptor and its effect on endothelial cell proliferation in infantile hemangioma.

BACKGROUND: Propranolol is the preferred treatment for problematic proliferating infantile hemangioma (IH) by targeting the renin-angiotensin system (RAS) expressed by IH endothelium. (Pro)renin receptor (PRR) is a major component of the RAS associated with the canonical wnt signaling pathway. We proposed that activation of PRR by renin causes proliferation of IH. METHODS: The expression of PRR in IH tissue samples was investigated using immunohistochemical (IHC) staining and NanoString analysis. NanoString analysis was also used to confirm transcriptional expression of PRR in CD34-sorted proliferating IH-derived primary cell lines. MTT assay was utilized to determine the effect of exogenous renin on the number of viable IH cells. RT-qPCR was used to determine the effect of renin on the stem cell gene expression. RESULTS: NanoString analysis and IHC staining confirmed transcriptional and translational expression of PRR, which was localized to the non-endothelial and the endothelial IH cell populations. MTT assay demonstrated an increased number of viable IH cells by administration of renin and the effect was negated by the wnt receptor blocker dickkopf-1. CONCLUSION: Our results present a model for renin-induced increased proliferation of IH cells through PRR acting via the wnt signaling pathway, which may account for accumulation of cells in IH during the proliferative phase of the tumor.

Expression of Cathepsins B, D, and G in WHO Grade I Meningioma.

Aim: We have recently demonstrated the presence of putative tumor stem cells (TSCs) in World Health Organization (WHO) grade I meningioma (MG) localized to the microvessels, which expresses components of the renin-angiotensin system (RAS). The RAS is known to be dysregulated and promotes tumorigenesis in many cancer types, including glioblastoma. Cathepsins B, D, and G are isoenzymes that catalyze the production of angiotensin peptides, hence providing bypass loops for the RAS. This study investigated the expression of cathepsins B, D, and G in WHO grade I MG in relation to the putative TSC population we have previously demonstrated. Methods: 3,3-Diaminobenzidine (DAB) immunohistochemical (IHC) staining with antibodies for cathepsins B, D, and G was performed on WHO grade I MG tissue samples from 10 patients. Three of the MG samples subjected to DAB IHC staining underwent immunofluorescence (IF) IHC staining to investigate co-expression of each of these cathepsins using combinations of smooth muscle actin (SMA) and embryonic stem cell marker OCT4. NanoString mRNA expression (n = 6) and Western blotting (WB; n = 5) analyses, and enzyme activity assays (EAAs; n = 3), were performed on snap-frozen WHO grade I MG tissue samples to confirm transcriptional activation, protein expression, and functional activity of these proteins, respectively. Results: DAB IHC staining demonstrated expression of cathepsins B, D, and G in all 10 MG samples. NanoString mRNA expression and WB analyses showed transcriptional activation and protein expression of all three cathepsins, although cathepsin G was expressed at low levels. EAAs demonstrated that cathepsin B and cathepsin D were functionally active. IF IHC staining illustrated localization of cathepsin B and cathepsin D to the endothelium and SMA+ pericyte layer of the microvessels, while cathepsin G was localized to cells scattered within the interstitium, away from the microvessels. Conclusion: Cathepsin B and cathepsin D, and to a lesser extent cathepsin G, are expressed in WHO grade I MG. Cathepsin B and cathepsin D are enzymatically active and are localized to the putative TSC population on the microvessels, whereas cathepsin G was localized to cells scattered within the interstitium, These results suggest the presence of bypass loops for the RAS, within WHO grade I MG.

Expression of Components of the Renin-Angiotensin System in Pyogenic Granuloma.

Background: There is a growing body of research demonstrating expression of the renin-angiotensin system (RAS) by a putative embryonic stem cell (ESC)-like population within vascular anomalies. This study investigated the expression of components of the RAS in relation to the putative ESC-like population within pyogenic granuloma (PG) that we have recently reported. Methods: PG samples from 14 patients were analyzed for the expression of components of the RAS: pro-renin receptor (PRR), angiotensin converting enzyme (ACE), angiotensin II receptor 1 (ATIIR1) and angiotensin II receptor 2 (ATIIR2), using 3,3-diaminobenzidine (DAB) immunohistochemical (IHC) staining. Immunofluorescence (IF) IHC staining was performed to localize these proteins on four of the PG samples. RT-qPCR was performed on two snap-frozen PG samples. Western blotting (WB) was performed on one snap-frozen PG sample and two PG-derived primary cell lines. Results: DAB IHC staining demonstrated the expression of ACE, PRR, ATIIR1, and ATIIR2 in all 14 PG tissue samples. RT-qPCR analysis confirmed abundant mRNA transcripts for PRR, ACE, AIITR1 and ATIIR2, relative to the housekeeping gene. WB confirmed the presence of PRR, ATIIR1, and ACE in the PG tissue sample, and PRR and ATIIR1, in the PG-derived primary cell lines. IF IHC staining demonstrated the expression of PRR, ACE, ATIIR1 on the primitive population that expressed NANOG and SOX2 on the ERG+ endothelium of the microvessels within PG. Conclusion: We have demonstrated the expression of PRR, ACE, and ATIIR1 by the putative the ESC-like population within PG.

Cerebral infarction after fractionated stereotactic radiation therapy of benign anterior skull base tumors.

BACKGROUND: The purpose of this study was to examine the occurrence of cerebral infarction (ischemic stroke), in a large combined cohort of patients with anterior skull base meningiomas, pituitary adenomas and craniopharyngiomas, after fractionated stereotactic radiation therapy (FSRT). MATERIAL AND METHODS: All patients, 18 years and older, with anterior skull base meningiomas, pituitary adenomas and craniopharyngiomas, treated with fractionated stereotactic radiation, in our center, from January 1999 to December 2015 were identified. In total 169 patients were included. The prescription dose to the tumor was 54 Gy for 164 patients (97%) and 46.0-52.2 Gy for 5 patients (3%). Cases of cerebral infarctions subsequent to FSRT were identified from the Danish National Patient Registry and verified with review of case notes. The rate of cerebral infarction after FSRT was compared to the rate in the general population with a one sample t-test after standardization for age and year. We explored if age, sex, disease type, radiation dose and dose per fraction was associated with increased risk of cerebral infarction using univariate Cox models. RESULTS: At a median follow-up of 9.3 years (range 0.1-16.5), 7 of the 169 patients (4.1%) developed a cerebral infarction, at a median 5.7 years (range 1.2-11.5) after FSRT. The mean cerebral infarction rate for the general population was 0.0035 and 0.0048 for the FSRT cohort (p = 0.423). Univariate cox models analysis showed that increasing age correlated significantly with the cerebral infarction risk, with a hazard ratio of 1.090 (p = 0.013). CONCLUSION: Increased risk of cerebral infarction after FSRT of anterior skull base tumors was associated with age, similar to the general population. Our study revealed that FSRT did not introduce an excess risk of cerebral infarction.

Expression of Embryonic Stem Cell Markers in Microcystic Lymphatic Malformation.

Aim: To investigate the expression of embryonic stem cell (ESC) markers in microcystic lymphatic malformation (mLM). Methods and Results: Cervicofacial mLM tissue samples from nine patients underwent 3,3'-diaminobenzidine (DAB) immunohistochemical (IHC) staining for ESC markers octamer-binding protein 4 (OCT4), homeobox protein NANOG, sex determining region Y-box 2 (SOX2), Krupple-like factor (KLF4), and proto-oncogene c-MYC. Transcriptional activation of these ESC markers was investigated using real-time polymerase chain reaction (RT-qPCR) and colorimetric in situ hybridization (CISH) on four and five of these mLM tissue samples, respectively. Immunofluorescence (IF) IHC staining was performed on three of these mLM tissue samples to investigate localization of these ESC markers. DAB and IF IHC staining demonstrated the expression of OCT4, SOX2, NANOG, KLF4, and c-MYC on the endothelium of lesional vessels with abundant expression of c-MYC and SOX2, which was also present on the cells within the stroma, in all nine mLM tissue samples. RT-qPCR and CISH confirmed transcriptional activation of all these ESC markers investigated. Conclusions: These findings suggest the presence of a primitive population on the endothelium of lesional vessels and the surrounding stroma in mLM. The abundant expression of the progenitor-associated markers SOX2 and c-MYC suggests that the majority are of progenitor phenotype with a small number of ESC-like cells.

Interpersonal Violence and Maxillofacial Fractures.

PURPOSE: Accident Compensation Corporation statistics shows that maxillofacial fracture affects 11,000 people with an approximate $90 million annual cost in New Zealand dollars (NZD). Previous studies have demonstrated interpersonal violence (IPV), road traffic accidents (RTAs), sports injury, and falls being the common causes of maxillofacial fracture. This study investigated the causes and associated alcohol and/or drug use and fracture patterns in patients presenting with maxillofacial fractures in the Wellington region. SUBJECTS AND METHODS: Demographic data of the patients, the cause of maxillofacial fracture and associated alcohol and/or drug use, and the fracture patterns were culled from our prospective maxillofacial fracture database at Hutt Hospital for a 5-year period from January 01, 2013, to December 31, 2017 and analyzed. RESULTS: A total of 1535 patients were referred with maxillofacial fractures during the study. 38% of the maxillofacial fractures were caused by IPV, followed by sports injury (24%), falls (24%), and RTA (6%), with 33.4% associated with alcohol and/or drug use. Males were six times more likely to present with IPV-related maxillofacial fractures, compared to females. The 16-30-year age group was the most prevalent in the IPV group with NZ Maori attributing to significantly more maxillofacial fractures compared to NZ European, 54.6% vs. 32.0% (P < 0.0001). CONCLUSIONS: IPV, especially involving alcohol and/or drug use, is the most common cause of maxillofacial fractures in the Wellington region, especially in NZ Maori males aged 16-30 years. Public health strategies are needed to decrease IPV as a cause of maxillofacial fractures.

Cancer stem cells within moderately differentiated head and neck cutaneous squamous cell carcinoma express components of the renin-angiotensin system.

PURPOSE: To investigate the expression of components of the renin-angiotensin system (RAS): pro-renin receptor (PRR), angiotensin converting enzyme (ACE), angiotensin II receptor 1 (ATIIR1) and angiotensin II receptor 2 (ATIIR2) by the cancer stem cell (CSC) subpopulations in moderately differentiated head and neck cutaneous squamous cell carcinoma (MDHNCSCC). METHODOLOGY: 3,3-Diaminobenzidine (DAB) immunohistochemical (IHC) staining for PRR, ACE, ATIIR1 and ATIIR2 was performed on formalin-fixed paraffin-embedded sections of ten MDHNCSCC tissue samples. Immunofluorescence (IF) IHC staining was used to localise components of the RAS. Western blotting (WB) and RT-qPCR were performed on snap-frozen MDHNCSCC tissue samples and MDHNCSCC-derived primary cell lines to investigate protein transcription expression of these proteins, respectively. RESULTS: DAB IHC staining demonstrated the presence of PRR, ACE, ATIIR1 and ATIIR2 in all ten MDHNCSCC tissue samples. IF IHC staining showed expression of PRR and ATIIR2 by the OCT4+ cells, and ACE and ATIIR1 by the SOX2+ cells, within the tumour nests (TNs) and the peritumoural stroma (PTS). PRR, ACE, ATIIR1 and ATIIR2 were expressed by the endothelium of the microvessels within the PTS. WB confirmed protein expression for PRR, ACE and ATIIR1 in MDHNCSCC tissue samples and MDHNCSCC-derived primary cell lines. RT-qPCR showed transcriptional activation of PRR, ACE, ATIIR1 and ATIIR2 in MDHNCSCC tissue samples; and PRR, ACE, ATIIR1 but not ATIIR2, in MDHNCSCC-derived primary cell lines. CONCLUSION: PRR, ACE, ATIIR1 and ATIIR2 are expressed by the CSC subpopulations within the TNs, the PTS, and the endothelium of the microvessels within the PTS, in MDHNCSCC.

Expression of Embryonic Stem Cell Markers on the Microvessels of WHO Grade I Meningioma.

Aim: The presence of cells within meningioma (MG) that express embryonic stem cell (ESC) markers has been previously reported. However, the precise location of these cells has yet to be determined. Methods: 3,3-Diaminobenzidine (DAB) immunohistochemical (IHC) staining was performed on 11 WHO grade I MG tissue samples for the expression of the ESC markers OCT4, NANOG, SOX2, KLF4 and c-MYC. Immunofluorescence (IF) IHC staining was performed to investigate the localization of each of these ESC markers. NanoString and colorimetric in situ hybridization (CISH) mRNA expression analyses were performed on six snap-frozen MG tissue samples to confirm transcriptional activation of these proteins, respectively. Results: DAB IHC staining demonstrated expression of OCT4, NANOG, SOX2, KLF4, and c-MYC within all 11 MG tissue samples. IF IHC staining demonstrated the expression of the ESC markers OCT4, NANOG, SOX2, KLF4, and c-MYC on both the endothelial and pericyte layers of the microvessels. NanoString and CISH mRNA analyses confirmed transcription activation of these ESC markers. Conclusion: This novel finding of the expression of all aforementioned ESC markers in WHO grade I MG infers the presence of a putative stem cells population which may give rise to MG.

Expression and Localization of Cathepsins B, D and G in Cancer Stem Cells in Liver Metastasis From Colon Adenocarcinoma.

AIM: We have previously identified and characterized cancer stem cell (CSC) subpopulations in liver metastasis from colon adenocarcinoma (LMCA). In this study we investigated the expression and localization of cathepsins B, D and G, in relation to these CSCs. METHODS: 3,3-Diaminobenzidine (DAB) immunohistochemical (IHC) staining for cathepsins B, D and G was performed on 4µm-thick formalin-fixed paraffin-embedded LMCA sections from nine patients. Immunofluorescence (IF) IHC staining was performed on three representative samples of LMCA from the original cohort of nine patients, to determine the localization of these cathepsins in relation to the CSC subpopulations. NanoString mRNA analysis and Western Blotting (WB) were used to examine the transcript and protein expression of these cathepsins, respectively. Enzyme activity assays were utilized to determine their functional activity. Data acquired from counting of cells staining positively of the cathepsins on the DAB IHC-stained slides and from Nanostring mRNA analysis were subjected to statistical analyses to determine significance. RESULTS: DAB IHC staining demonstrated expression of cathepsins B, D and G within LMCA. IF IHC staining demonstrated the expression of both cathepsin B and cathepsin D by the OCT4- cells within the tumor nests and the OCT4+ CSC subpopulation within the peritumoral stroma. NanoString mRNA analysis showed significantly greater transcript expression of cathepsin B and cathepsin D, compared to cathepsin G. WB confirmed expression of cathepsin B and cathepsin D proteins, while cathepsin G was below detectable levels. Enzyme activity assays showed functional activity of cathepsin B and cathepsin D. CONCLUSION: Our study demonstrated novel finding of the expression of cathepsin B, cathepsin D, and possibly cathepsin G by the putative CSC subpopulations within LMCA.

Expression and Localization of Cathepsins B, D, and G in Dupuytren's Disease.

BACKGROUND: The pathogenesis of Dupuytren's disease (DD) remains unclear. An embryonic stem cell (ESC)-like population in the endothelium of the microvessels around tissues that expresses components of the renin-angiotensin system (RAS) has been reported. This study investigated if this primitive population expresses cathepsins B, D, and G, that contribute to RAS bypass loops. METHODS: 3,3-Diaminobenzidine immunohistochemical (IHC) staining for cathepsins B, D, and G was performed on sections of formalin-fixed paraffin-embedded DD cords (n = 10) and nodules (n = 10). Immunofluorescence IHC staining was utilized to demonstrate co-expression of these cathepsins with ESC markers. Protein and gene expression of these cathepsins was investigated in snap-frozen DD cords (n = 3) and nodules (n = 3) by Western blotting and NanoString analysis, respectively. Enzymatic activity of these cathepsins was investigated by enzymatic activity assays. RESULTS: 3,3-Diaminobenzidine IHC staining demonstrated expression of cathepsins B, D, and G in DD cords and nodules. Gene expression of cathepsins B, D, and G was confirmed by NanoString analysis. Western blotting confirmed expression of cathepsins B and D, but not cathepsin G. Immunofluorescent IHC staining demonstrated high abundance of cathepsins B and D on the OCT4+/angiotensin converting enzyme+ endothelium and the smooth muscle layer of the microvessels. Cathepsin G was localized to trypase+ cells within the stroma in DD cords and nodules with limited expression on the microvessels. Enzyme activity assays demonstrated functional activity of cathepsins B and D. CONCLUSIONS: Cathepsins B, D, and G were expressed in the DD tissues, with cathepsins B and D localized to the primitive population in the endothelium of the microvessels, whereas cathepsin G was localized to phenotypic mast cells, suggesting the presence of bypass loops for the RAS.

Expression and Localization of Cathepsins B, D, and G in Two Cancer Stem Cell Subpopulations in Moderately Differentiated Oral Tongue Squamous Cell Carcinoma.

AIM: We have previously demonstrated the putative presence of two cancer stem cell (CSC) subpopulations within moderately differentiated oral tongue squamous cell carcinoma (MDOTSCC), which express components of the renin-angiotensin system (RAS). In this study, we investigated the expression and localization of cathepsins B, D, and G in relation to these CSC subpopulations within MDOTSCC. METHODS: 3,3-Diaminobenzidine (DAB) and immunofluorescent (IF) immunohistochemical (IHC) staining was performed on MDOTSCC samples to determine the expression and localization of cathepsins B, D, and G in relation to the CSC subpopulations. NanoString mRNA analysis and colorimetric in situ hybridization (CISH) were used to study their transcripts expression. Enzyme activity assays were performed to determine the activity of these cathepsins in MDOTSCC. RESULTS: IHC staining demonstrated expression of cathepsins B, D, and G in MDOTSCC. Cathepsins B and D were localized to CSCs within the tumor nests, while cathepsin B was localized to the CSCs within the peri-tumoral stroma, and cathepsin G was localized to the tryptase+ phenotypic mast cells within the peri-tumoral stroma. NanoString and CISH mRNA analyses confirmed transcription activation of cathepsins B, D, and G. Enzyme activity assays confirmed active cathepsins B and D, but not cathepsin G. CONCLUSION: The presence of cathepsins B and D on the CSCs and cathspsin G on the phenotypic mast cells suggest the presence of bypass loops for the RAS which may be a potential novel therapeutic target for MDOTSCC.

Expression of Cathepsins B, D, and G in Isocitrate Dehydrogenase-Wildtype Glioblastoma.

AIM: To investigate the expression of cathepsins B, D, and G, in relation to the cancer stem cell (CSC) subpopulations, we have previously characterized within isocitrate dehydogenase (IDH)-wildtype glioblastoma (IDHWGB). METHODS: 3,3-Diaminobezidine (DAB) immunohistochemical (IHC) staining for cathepsins B, D, and G, was performed on 4µm-thick formalin-fixed paraffin-embedded IDHWGB samples obtained from six patients. Two representative DHWGB samples from the original cohort of patients were selected for immunofluorescent (IF) IHC staining, to identify the localization of the cathepsins in relation to the CSC subpopulations. NanoString gene expression analysis and colorimetric in situ hybridization (CISH) were conducted to investigate the transcriptional activation of genes encoding for cathepsins B, D, and G. Data obtained from cell counting of DAB IHC-stained slides and from NanoString analysis were subjected to statistical analyses to determine significance. RESULTS: Cathepsin B and cathepsin D were detected in IDHWGB by DAB IHC staining. IF IHC staining demonstrated the expression of both cathepsin B and cathepsin D by the OCT4+ and SALL4+ CSC subpopulations. NanoString gene analysis and CISH confirmed the abundant transcript expression of these cathepsins. The transcriptional and translational expressions of cathepsin G were minimal and were confined to cells within the microvasculature. CONCLUSION: This study demonstrated the expression of cathepsin B and cathepsin D but not cathepsin G within the CSC subpopulations of IDHWGB at both the transcriptional and translational level. Cathepsin G was expressed at low levels and was not localized to the CSC population of IDHWGB. The novel finding of cathepsin B and cathepsin D in IDHWGB suggests the presence of bypass loops for the renin-angiotensin system, which may facilitate the production of angiotensin peptides. Elucidating the precise role of these cathepsins may lead to better understanding and more effective treatment of this aggressive tumor.

Characterization of Cancer Stem Cells in Colon Adenocarcinoma Metastasis to the Liver.

BACKGROUND: Fifty percent of colorectal cancer (CRC) patients develop liver metastasis. This study identified and characterized cancer stem cells (CSCs) within colon adenocarcinoma metastasis to the liver (CAML). METHODS: 3,3-Diaminobenzidine immunohistochemical (IHC) staining was performed on nine CAML samples for embryonic stem cell (ESC) markers OCT4, SOX2, NANOG, c-Myc, and KLF4. Immunofluorescence (IF) IHC staining was performed to investigate coexpression of two markers. NanoString mRNA expression analysis and colorimetric in situ hybridization (CISH) were performed on four snap-frozen CAML tissue samples for transcript expression of these ESC markers. Cells stained positively and negatively for each marker by IHC and CISH staining were counted and analyzed. RESULTS: 3,3-Diaminobenzidine IHC staining, and NanoString and CISH mRNA analyses demonstrated the expression of OCT4, SOX2, NANOG, c-Myc, and KLF4 within in all nine CAML samples, except for SOX2 which was below detectable levels on NanoString mRNA analysis. IF IHC staining showed the presence of a SOX2+/NANOG+/KLF4+/c-Myc+/OCT- CSC subpopulation within the tumor nests, and a SOX2+/NANOG+/KLF4+/c-Myc+/OCT4- CSC subpopulation and a SOX2+/NANOG+/KLF4+/c-Myc+/OCT4+ CSC subpopulation within the peritumoral stroma. CONCLUSION: The novel finding of three CSC subpopulations within CAML provides insights into the biology of CRC.

Neuropeptide Y receptor 1 is expressed by B and T lymphocytes and mast cells in infantile haemangiomas.

AIM: We investigated the expression of neuropeptide Y (NPY), NPY receptor 1 (NPYR1) and NPY receptor 2 (NPYR2) in infantile haemangiomas (IHs). METHODS: Immunohistochemical (IHC) staining was performed on proliferating IHs from six patients aged 4-13 (mean 8.7) months and involuted IHs from six patients aged 5-59 (mean 18.7) years, for the expression of NPY, NPYR1 and NPYR2. Protein and messenger ribonucleic acid expression corresponding to these proteins was investigated by Western blotting and NanoString analysis, respectively. RESULTS: IHC staining, Western blotting and NanoString analysis demonstrated the presence of NPYR1, but not NPYR2, within proliferating and involuted IHs. IHC staining showed NPYR1 was expressed by B and T lymphocytes expressing CD45 and mast cells expressing tryptase. IHC staining demonstrated the presence of NPY on the NPYR1+ cells, but it was not detected by Western blotting or NanoString analysis. CONCLUSION: NPYR1, but not NPYR2, was present in IHs. The localisation of NPYR1 to B and T lymphocytes and mast cells suggests its role in the biology of IHs. The demonstration of NPY on the NPYR1+ cells, without active transcription, suggests that NPY was not being produced within IHs.