Cancer stem cells (CSCs) have been identified in many cancer types including primary head and neck cutaneous squamous cell carcinoma (HNcSCC). This study aimed to identify and characterize CSCs in metastatic HNcSCC (mHNcSCC). Immunohistochemical staining performed on mHNcSCC samples from 15 patients demonstrated expression of the induced pluripotent stem cell (iPSC) markers OCT4, SOX2, NANOG, KLF4, and c-MYC in all 15 samples. In situ hybridization and RT-qPCR performed on four of these mHNcSCC tissue samples confirmed transcript expression of all five iPSC markers. Immunofluorescence staining performed on three of these mHNcSCC samples demonstrated expression of c-MYC on cells within the tumor nests (TNs) and the peri-tumoral stroma (PTS) that also expressed KLF4. OCT4 was expressed on the SOX2+/NANOG+/KLF4+ cells within the TNs, and the SOX2+/NANOG+/KLF4+ cells within the PTS. RT-qPCR demonstrated transcript expression of all five iPSC markers in all three mHNcSCC-derived primary cell lines, except for SOX2 in one cell line. Western blotting showed the presence of SOX2, KLF4, and c-MYC but not OCT4 and NANOG in the three mHNcSCC-derived primary cell lines. All three cell lines formed tumorspheres, at the first passage. We demonstrated an OCT4+/NANOG+/SOX2+/KLF4+/c-MYC+ CSC subpopulation and an OCT4+/NANOG-/SOX2+/KLF4+/c-MYC+ subpopulation within the TNs, and an OCT4+/NANOG+/SOX2+/KLF4+/c-MYC+ subpopulation within the PTS of mHNcSCC.

Objectives: Arteriovenous malformation (AVM) consists of a nidus with poorly formed low-resistance vessels in place of a functional capillary network. The role of somatic mutations in embryonic stem cells (ESCs) and vascular anomalies and the presence of primitive populations in vascular anomalies led us to investigate the presence of a primitive population in extracranial AVM.Methods: Extracranial AVM tissue samples from 12 patients were stained for stemness-associated markers OCT4, SOX2, NANOG, KLF4, and c-MYC using immunohistochemical staining. In situ hybridization (ISH) was performed on six tissue samples to determine transcript expression. Western blotting and RT-qPCR were performed on two AVM-derived primary cell lines to determine protein and transcript expression of these markers, respectively. Immunofluorescence staining was performed on two tissue samples to investigate marker co-localization.Results: Immunohistochemical staining demonstrated the expression of OCT4, SOX2, KLF4, and c-MYC on the endothelium and media of lesional vessels and cells within the stroma of the nidus in all 12 AVM tissue samples. ISH and RT-qPCR confirmed transcript expression of all five markers. Western blotting showed protein expression of all markers except NANOG. Immunofluorescence staining demonstrated an OCT4+/SOX2+/KLF4+/c-MYC+ population within the endothelium and media of the lesional vessels and cells within the stroma of the AVM nidus.Conclusions: Our findings may suggest the presence of a primitive population within the AVM nidus. Further investigation may lead to novel therapeutic targeting of this population.

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Objective: To identify and characterize an embryonic stem cell (ESC)-like population within hypertrophic port-wine stain (HPWS). Methods: HPWS tissue samples from 15 patients underwent immunohistochemical staining for induced pluripotent stem cell (iPSC) markers OCT4, SOX2, NANOG, KLF4, and c-MYC. Immunofluorescence staining was performed on 2 of these tissue samples to investigate colocalization of these markers. In situ hybridization and reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) were performed on 6 of the HPWS samples to investigate transcript expression of these iPSC markers. Western blotting and RT-qPCR were performed on 3 HPWS-derived primary cell lines, to determine protein and transcript expression of these markers, respectively. Results: Immunohistochemical staining demonstrated expression of OCT4, SOX2, KLF4, and c-MYC but not NANOG on the endothelium and media of lesional vessels and on cells within the stroma in all 15 HPWS tissue samples. Immunofluorescence staining showed the presence of an OCT4+/SOX2+/NANOG-/KLF4+/c-MYC+ ESC-like subpopulation within the endothelium and media of the lesional vessels, and cells within the stroma of HPWS. In situ hybridization detected OCT4, SOX2, KLF4, and c-MYC transcripts in all 6 HPWS tissue samples. RT-qPCR demonstrated transcripts of all 5 iPSC markers in the HPWS tissue samples and in the HPWS-derived primary cell lines, which expressed OCT4, SOX2, KLF4, and c-MYC but not NANOG proteins by Western blotting. Conclusion: This study demonstrated an OCT4+/SOX2+/NANOG-/KLF4+/c-MYC+ ESC-like population within the endothelium and media of the lesional vessels and the cells within the stroma of HPWS.

Objectives: To investigate the presence of components of the renin-angiotensin system (RAS) on the embryonic stem cell (ESC)–like population in microcystic lymphatic malformation (mLM). Methods: mLM tissue samples from 18 patients underwent immunohistochemical staining for RAS components including angiotensinogen, renin, prorenin receptor (PRR), angiotensin-converting enzyme (ACE), ACE2, and angiotensin II receptor 2 (AT2R). Snap-frozen mLM tissues from 6 of the patients were used to confirm protein expression by western blotting for angiotensinogen, PRR, ACE, ACE2, and AT2R. Reverse transcription quantitative polymerase chain reaction was used to detect transcript expression of angiotensinogen, renin, PRR, ACE, ACE2, AT1R, and AT2R in 5 of the mLM tissue samples. Results: Immunohistochemical staining demonstrated expression of AT2R in all, and PRR in 1, while angiotensinogen, renin, ACE, and ACE2 were not observed in any of the 18 mLM samples. Western blotting showed expression of angiotensinogen, PRR, and ACE, but not ACE2 or AT2R in all 6 mLM tissue samples. Reverse transcription quantitative polymerase chain reaction demonstrated transcript expression of angiotensinogen, PRR, ACE, and ACE2 in all, AT1R in 4, AT2R in 2, and renin in 1 of the 5 mLM tissue samples. Immunofluorescence dual-staining in 2 mLM tissue samples demonstrated expression of AT2R on the OCT4+ cells. Conclusion: This study shows expression of angiotensinogen, PRR, ACE. ACE2, AT1R, and AT2R transcripts and AT2R protein, in mLM tissue samples, with AT2R localizing to the OCT4+ ESC-like population. This suggests the ESC-like population may be a novel therapeutic target by modulation of the RAS.