Transglutaminase 3 regulates cutaneous squamous carcinoma differentiation and inhibits progression via PI3K-AKT signaling pathway-mediated Keratin 14 degradation.

Cutaneous squamous carcinoma is the second most common epithelial malignancy, associated with significant morbidity, mortality, and economic burden. However, the mechanisms underlying cSCC remain poorly understood. In this study, we identified TGM3 as a novel cSCC tumor suppressor that acts via the PI3K-AKT axis. RT-qPCR, IHC and western blotting were employed to assess TGM3 levels. TGM3-overexpression/knockdown cSCC cell lines were utilized to detect TGM3's impact on epithelial differentiation as well as tumor cell proliferation, migration, and invasion in vitro. Additionally, subcutaneous xenograft tumor models were employed to examine the effect of TGM3 knockdown on tumor growth in vivo. Finally, molecular and biochemical approaches were employed to gain insight into the tumor-suppressing mechanisms of TGM3. TGM3 expression was increased in well-differentiated cSCC tumors, whereas it was decreased in poor-differentiated cSCC tumors. Loss of TGM3 is associated with poor differentiation and a high recurrence rate in patients with cSCC. TGM3 exhibited tumor-suppressing activity by regulating cell proliferation, migration, and invasion both in vitro and in vivo. As a novel cSCC tumor differentiation marker, TGM3 expression was positively correlated with cell differentiation. In addition, our results demonstrated an interaction between TGM3 and KRT14 that aids in the degradation of KRT14. TGM3 deficiency disrupts keratinocytes differentiation, and ultimately leads to tumorigenesis. Furthermore, RNA-sequence analysis revealed that loss of TGM3 enhanced EMT via the PI3K-AKT signaling pathway. Deguelin, a PI3K-AKT inhibitor, blocked cSCC tumor growth induced by TGM3 knockdown in vivo. Taken together, TGM3 inhibits cSCC tumor growth via PI3K-AKT signaling, which could also serve as a tumor differentiation marker and a potential therapeutic target for cSCC. Proposed model depicted the mechanism by which TGM3 suppress cSCC development. TGM3 reduces the phosphorylation level of AKT and degrades KRT14. In the epithelial cell layer, TGM3 exhibits a characteristic pattern of increasing expression from bottom to top, while KRT14 and pAKT are the opposite. Loss of TGM3 leads to reduced degradation of KRT14 and activation of pAKT, disrupting keratinocyte differentiation, and eventually resulting in the occurrence of low-differentiated cSCC.

Primary cutaneous anaplastic large-cell lymphoma resembling infratemporal space infection: a case report.

BACKGROUND: Primary cutaneous anaplastic large-cell lymphoma (PC-ALCL) is a rare T-cell lymphoma belonging to the CD30 + T-cell lymphoproliferative disorders. The case of PC-ALCL in the temporal region is exceedingly rare. Herein, we report a case of PC-ALCL involving the temporal region mimicking infratemporal space infection. CASE PRESENTATION: A 78-year-old woman presented to maxillofacial surgery service with a 6-month history of swelling and pain in the left side of her face. Laboratory investigations found an elevated C-reactive protein (CRP). Imaging findings showed enlarged lymph nodes and extensive thickening of subcutaneous tissue of the left temples. Based on these findings, the infratemporal space infection was suspected initially. The patient underwent incision and drainage, and we unexpectedly found no pus in the lesion area. Incisional biopsy showed necrosis and extensive involvement of the left temples by a diffuse infiltrate containing large, atypical cells. The tumor cells were positive for CD30, CD3, Ki67. They were negative for ALK (SP8), CD5, CD8, CD20 and PAX5. After considering these findings, a diagnosis of PC-ALCL was rendered. The patient was admitted to the lymphoma department for systemic chemotherapy and no relapse occurred during a follow-up period of six months. CONCLUSIONS: This report suggests that if there are suspicious intraoperative manifestations, carrying out a biopsy simultaneously, using Hematoxylin and eosin (HE) staining, and a comprehensive Immunohistochemistry (IHC) panel are essential to diagnosing PC-ALCL to prevent misdiagnosis.

Spectrum of malignant and premalignant skin lesions in 505 adult subjects at risk of skin cancers.

Patients at risk of skin cancers can develop varying types of cutaneous malignancies. However, some subjects may develop only one type of lesion. In this cross-sectional study, the spectrum of premalignant (PM) and malignant skin lesions and their risk factors were studied. Therefore, 505 adult subjects (aged 21-79 years, 256 males and 249 females, 96 with immunosuppression) at risk of any type of skin cancer were examined for cutaneous malignancies, nevi, actinic keratoses, photodamage, and possible risk factors. First, 12 different groups were identified with a varying set of PM and/or malignant skin lesions. Next, 5 larger groups were formed from them: basal cell carcinoma (BCC) only, malignant melanoma (MM) only, squamous cell carcinoma (SCC) and/or PM, BCC + SCC and/or PM, and MM + keratinocyte carcinoma (KC) and/or PM. The groups with BCC or MM only were younger and showed less photodamage than the mixed groups, while SCC/PM showed similarity with them. In logistic regression analyses, the platelet-to-lymphocyte ratio was associated with an increased risk of concomitant KC (OR 1.028, p = 0.023) or SCC/PM (OR 1.009, p = 0.047) in subjects with MM or BCC, respectively. Actinic keratoses produced ORs 0.246-0.252 (p = 0.008-0.020) for BCC in subjects with SCC/PM. Interestingly, atypical mole syndrome decreased the risk of SCC/PM in subjects with BCC (OR 0.092, p = 0.001). Advanced age was a significant risk factor for an additional type of lesion in all 3 comparisons (ORs 1.088-1.388, p = 0.001). In conclusion, even though there are numerous patients with only one lesion type, advancing age may determine the final lesion multiplicity.

Unraveling lipid metabolism reprogramming for overcoming drug resistance in melanoma.

Cutaneous melanoma is the deadliest form of skin cancer, and its incidence is continuing to increase worldwide in the last decades. Traditional therapies for melanoma can easily cause drug resistance, thus the treatment of melanoma remains a challenge. Various studies have focused on reversing the drug resistance. As tumors grow and progress, cancer cells face a constantly changing microenvironment made up of different nutrients, metabolites, and cell types. Multiple studies have shown that metabolic reprogramming of cancer is not static, but a highly dynamic process. There is a growing interest in exploring the relationship between melanoma andmetabolic reprogramming, one of which may belipid metabolism. This review frames the recent research progresses on lipid metabolism in melanoma.In addition, we emphasize the dynamic ability of metabolism during tumorigenesis as a target for improving response to different therapies and for overcoming drug resistance in melanoma.

Skin Protection by Carotenoid Pigments.

Sunlight, despite its benefits, can pose a threat to the skin, which is a natural protective barrier. Phototoxicity caused by overexposure, especially to ultraviolet radiation (UVR), results in burns, accelerates photoaging, and causes skin cancer formation. Natural substances of plant origin, i.e., polyphenols, flavonoids, and photosynthetic pigments, can protect the skin against the effects of radiation, acting not only as photoprotectors like natural filters but as antioxidant and anti-inflammatory remedies, alleviating the effects of photodamage to the skin. Plant-based formulations are gaining popularity as an attractive alternative to synthetic filters. Over the past 20 years, a large number of studies have been published to assess the photoprotective effects of natural plant products, primarily through their antioxidant, antimutagenic, and anti-immunosuppressive activities. This review selects the most important data on skin photodamage and photoprotective efficacy of selected plant carotenoid representatives from in vivo studies on animal models and humans, as well as in vitro experiments performed on fibroblast and keratinocyte cell lines. Recent research on carotenoids associated with lipid nanoparticles, nanoemulsions, liposomes, and micelles is reviewed. The focus was on collecting those nanomaterials that serve to improve the bioavailability and stability of carotenoids as natural antioxidants with photoprotective activity.

Merkel cell polyomavirus-specific and CD39+CLA+ CD8 T cells as blood-based predictive biomarkers for PD-1 blockade in Merkel cell carcinoma.

Merkel cell carcinoma is a skin cancer often driven by Merkel cell polyomavirus (MCPyV) with high rates of response to anti-PD-1 therapy despite low mutational burden. MCPyV-specific CD8 T cells are implicated in anti-PD-1-associated immune responses and provide a means to directly study tumor-specific T cell responses to treatment. Using mass cytometry and combinatorial tetramer staining, we find that baseline frequencies of blood MCPyV-specific cells correlated with response and survival. Frequencies of these cells decrease markedly during response to therapy. Phenotypes of MCPyV-specific CD8 T cells have distinct expression patterns of CD39, cutaneous lymphocyte-associated antigen (CLA), and CD103. Correspondingly, overall bulk CD39+CLA+ CD8 T cell frequencies in blood correlate with MCPyV-specific cell frequencies and similarly predicted favorable clinical outcomes. Conversely, frequencies of CD39+CD103+ CD8 T cells are associated with tumor burden and worse outcomes. These cell subsets can be useful as biomarkers and to isolate blood-derived tumor-specific T cells.

CSDE1 Intracellular Distribution as a Biomarker of Melanoma Prognosis.

RNA-binding proteins are emerging as critical modulators of oncogenic cell transformation, malignancy and therapy resistance. We have previously found that the RNA-binding protein Cold Shock Domain containing protein E1 (CSDE1) promotes invasion and metastasis of melanoma, the deadliest form of skin cancer and also a highly heterogeneous disease in need of predictive biomarkers and druggable targets. Here, we design a monoclonal antibody useful for IHC in the clinical setting and use it to evaluate the prognosis potential of CSDE1 in an exploratory cohort of 149 whole tissue sections including benign nevi and primary tumors and metastasis from melanoma patients. Contrary to expectations for an oncoprotein, we observed a global decrease in CSDE1 levels with increasing malignancy. However, the CSDE1 cytoplasmic/nuclear ratio exhibited a positive correlation with adverse clinical features of primary tumors and emerged as a robust indicator of progression free survival in cutaneous melanoma, highlighting the potential of CSDE1 as a biomarker of prognosis. Our findings provide a novel feature for prognosis assessment and highlight the intricacies of RNA-binding protein dynamics in cancer progression.

Upregulation of IL4-induced gene 1 enzyme by B2 cells during melanoma progression impairs their antitumor properties.

B cells present in human cutaneous melanoma have been associated with protective or detrimental effects on disease progression according to their phenotype. By using the RET model of spontaneous melanoma and adoptive transfer of B16 melanoma cells, we show that immature and follicular B2 (B2-FO) cells exert a protective effect on melanoma progression by promoting the generation of effector memory T cells and limiting the recruitment of polymorphonuclear myeloid-derived suppressor cells. Unfortunately, this beneficial effect progressively wanes as a consequence of enhanced expression of the IL4-induced gene 1 (IL4I1) enzyme by immature B cells and B2-FO cells. Endogenous IL4I1 selectively decreases CXCR5 expression in splenic immature B cells, subverting their trafficking to primary tumors and enhancing the production of IL-10 by B2 cells, thereby promoting an immunosuppressive microenvironment. Accordingly, B2 cells from RET IL4I1KO mice more efficiently controlled B16 melanoma growth than B2 cells from IL4I1-competent RET mice. Collectively, immature B cells and B2-FO cells are key actors in the control of melanoma growth, but their mobility and functions are differently impaired by IL4I1 overexpression during melanoma progression. Thus, our present data strongly urge us to associate an IL4I1 antagonist with current immunotherapy to improve the treatment of metastatic melanoma.

Role of asprosin and meteorin-like peptide in progression of actinic keratosis to squamous cell carcinoma.

Squamous cell carcinoma (SCC) often develops from an underlying premalignant lesion. Factors that affect the progression of actinic keratosis (AK) to invasive SCC are not fully known. Asprosin (ASP) and meteorin-like peptide (METRNL) are adipokines that are involved primarily in glucose metabolism. We investigated the expression of ASP and METRNL in AK and SCC to evaluate the role of these adipokines in the development of SCC. We used 15 SCC specimens, 12 AK specimens and 12 healthy control skin specimens. ASP and METRNL protein expression in tumor and surrounding tissue was evaluated using immunohistochemistry. ASP expression in tumor tissue was significantly greater in the SCC group than in the control and AK groups, but it did not differ significantly between the AK and control groups. A positive correlation was observed for both ASP and METRNL expressions between tumor tissue and adjacent epidermis, hair follicles, sebaceous gland, eccrine gland, inflammatory cells and vascular structures. ASP and METRNL may exert pro-tumor effects toward development of invasive SCC. The expression intensity of ASP and METRNL can be used as a biomarker of risk of progression to SCC.

Diagnostic management of blastic plasmacytoid dendritic cell neoplasm (BPDCN) in close interaction with therapeutic considerations.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN), a rare malignancy derived from plasmacytoid dendritic cells, can mimic both acute leukemia and aggressive T-cell lymphoma. Therapy of this highly aggressive hematological disease should be initiated as soon as possible, especially in light of novel targeted therapies that have become available. However, differential diagnosis of BPDCN remains challenging. This retrospective study aimed to highlight the challenges to timely diagnoses of BPDCN. We documented the diagnostic and clinical features of 43 BPDCN patients diagnosed at five academic hospitals from 2001-2022. The frequency of BPDCN diagnosis compared to AML was 1:197 cases. The median interval from the first documented clinical manifestation to diagnosis of BPDCN was 3 months. Skin (65%) followed by bone marrow (51%) and blood (45%) involvement represented the most common sites. Immunophenotyping revealed CD4 + , CD45 + , CD56 + , CD123 + , HLA-DR + , and TCL-1 + as the most common surface markers. Overall, 86% (e.g. CD33) and 83% (e.g., CD7) showed co-expression of myeloid and T-cell markers, respectively. In the median, we detected five genomic alterations per case including mutational subtypes typically involved in AML: DNA methylation (70%), signal transduction (46%), splicing factors (38%), chromatin modification (32%), transcription factors (32%), and RAS pathway (30%), respectively. The contribution of patients (30%) proceeding to any form of upfront stem cell transplantation (SCT; autologous or allogeneic) was almost equal resulting in beneficial overall survival rates in those undergoing allogeneic SCT (p = 0.0001). BPDCN is a rare and challenging entity sharing various typical characteristics of other hematological diseases. Comprehensive diagnostics should be initiated timely to ensure appropriate treatment strategies.

Insights into the structure-activity relationship of pyrimidine-sulfonamide analogues for targeting BRAF V600E protein.

B-rapidly accelerated fibrosarcoma (BRAF) V600E plays a crucial role in the progression of cutaneous melanoma. Core structures of BRAF V600E inhibitors are based on pyrimidine-sulfonamide scaffolds. Exploring the QSAR of these structures can improve our understanding of BRAF V600E inhibitor drug design. This study utilized machine learning-based QSAR to elucidate chemical substructures of pyrimidine-sulfonamide analogues that correlated to the BRAF V600E inhibitory activity. The findings indicate that the support vector regression (SVR) combined with 15 fingerprints achieved the highest statistical performances in terms of goodness-of-fit, robustness, and predictability. Nine key fingerprints from pyrimidine-sulfonamide analogues were identified to exert the BRAF V600E inhibitory activity. These key fingerprints were validated using network-based activity cliff landscape and molecular docking. Together, the developed algorithm can serve as a screening tool for designing BRAF V600E inhibitors. To further utilize this model, we deployed our developed algorithm at https://qsarlabs.com/#braf.

Synergistic inhibitory actions of resveratrol, epigallocatechin-3-gallate, and diallyl trisulfide against skin cancer cell line A431 through mitochondrial caspase dependent pathway: a combinational drug approach.

The harmful effect of chemotherapeutic side effects has paid a way to discover a novel with curative way for skin cancer treatment. Skin cancer prevention is more viable with the use of combination of bioactive agents than using of single bioactive compounds. Present work was demonstrated to evaluate the interaction of Resveratrol (Res), Epigallocatechin-3-gallate (EGCG), and diallyl trisulfide (DATS) with each other as a binary combination on A431 cells. Nuclear fragmentation analysis of combination of bioactive agents using DAPI analysis, detection of apoptosis, analysis of cell cycle, ROS assay, antimigration assays, and western blotting were implemented to study the combination of bioactive compounds on A431 cell line. Among the selected combination EGCG + DATS had a synergetic effect reducing cellular migration, increased intercellular reactive oxygen species generation, condensation, cell phagocytosis induced by phosphatidylserine externalization, rise in sub-G1 DNA content, and S-phase were cell cycle arrest. The combinations EGCG + DATS induced apoptotic proteins in A431 cells by upregulation of proapoptotic Bax and Bad proteins, a downmodulation of anti-apoptotic proteins Bcl2 and caspases (caspase-3, and -9) activity got triggered by intrinsic pathway. The combination of EGCG + DATS showed good anticancer potential against A431 skin cancer cell line via the mitochondrial caspase dependent pathway with very strong synergism. This finding will help to produce a novel combination/chemoprevention using dietary bioactive agents (EGCG + DATS) for the treatment of skin cancer after clinical trial.

Screening the critical protein subnetwork to delineate potential mechanisms and protective agents associated with arsenic-induced cutaneous squamous cell carcinoma: A toxicogenomic study.

Recent studies show that complex mechanisms are involved in arsenic-induced malignant transformation of cells. This study aimed to decipher molecular mechanisms associated with arsenic-induced cutaneous squamous cell carcinoma (cSCC) and suggest potential protective factors. RNA-seq-based differentially expressed genes between arsenic-exposed human keratinocytes (HaCaT) and controls were used to construct a protein-protein interaction (PPI) network and discover critical subnetwork-based mechanisms. Protective compounds against arsenic toxicity were determined and their target interactions in the core sub-network were identified by the comparative toxicogenomic database (CTD). The binding affinity between the effective factor and target was calculated by molecular docking. A total of 15 key proteins were screened out as critical arsenic-responsive subnetwork (FN1, IL-1A, CCN2, PECAM1, FGF5, EDN1, FGF1, PXDN, DNAJB9, XBP1, ERN1, PDIA4, DNAJB11, FOS, PDIA6) and 7 effective protective agents were identified (folic acid, quercetin, zinc, acetylcysteine, methionine, catechin, selenium). The GeneMANIA predicted detailed interactions of the subnetwork and revealed terms related to unfolded protein response as the main processes. FN1, IL1A and CCN2, as top significant genes, had good docking affinity with folic acid and quercetin, as selected key compounds. Integration of gene expression and protein-protein interaction related to arsenic exposure in cSCC explored the potential mechanisms and protective agents.

Radix Tetrastigma Hemsleyani Flavone represses cutaneous squamous cell carcinoma via Janus kinase/signal transducer and activator of transcription 3 pathway inactivation.

Cutaneous squamous cell carcinoma (CSCC) is the second most common malignant skin tumor and significantly affects patients' quality of life and health. The Janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3) pathway activation is involved in CSCC development. Radix Tetrastigma hemsleyani flavone (RTHF) is an active Radix Tetrastigma extract (RTE), which was recently reported to have promising inhibitory effects on CSCC. However, the underlying functional mechanisms of this inhibition remain unknown. In the present study, A431 cells or SCL-1 cells were incubated with 1, 5, and 10 mg/mL RTHF for 48 h, respectively. A significantly increased wound closure rate, decreased number of migrated and invaded cells, decreased colony number, and elevated apoptotic rate were observed after treatment with 1, 5, and 10 mg/mL RTHF. Furthermore, after incubation with RTHF, p-JAK1/JAK1, p-JAK2/JAK2, and p-STAT3/STAT3 levels were drastically reduced. An A431 xenograft model was constructed, followed by oral administration of 15, 30, or 60 mg/kg RTHF for 21 consecutive days. A significantly lower increase in tumor volume and reduced tumor weight were observed in all RTHF-treated groups. In addition, JAK/STAT3 signaling was drastically repressed in tumor tissues. Collectively, RTHF inhibited CSCC progression, which may be associated with JAK/STAT3 pathway inactivation.

Mosaic BRAF Fusions Are a Recurrent Cause of Congenital Melanocytic Nevi Targetable by MAPK Pathway Inhibition.

Among children with multiple congenital melanocytic nevi, 25% have no established genetic cause, of whom many develop a hyperproliferative and severely pruritic phenotype resistant to treatment. Gene fusions have been reported in individual cases of congenital melanocytic nevi. We studied 169 patients with congenital melanocytic nevi in this study, 38 of whom were double wild type for pathogenic NRAS/BRAF variants. Nineteen of these 38 patients had sufficient tissue to undergo RNA sequencing, which revealed mosaic BRAF fusions in 11 of 19 patients and mosaic RAF1 fusions in 1 of 19. Recurrently, fusions involved the loss of the 5´ regulatory domain of BRAF or RAF1 but preserved the kinase domain. We validated all cases and detected the fusions in two separate nevi in 5 of 12 patients, confirming clonality. The absence of the fusion in blood in 8 of 12 patients indicated mosaicism. Primary culture of BRAF-fusion nevus cells from 3 of 12 patients demonstrated highly increased MAPK activation, despite only mildly increased BRAF expression, suggesting additional mechanisms of kinase activation. Trametinib quenched MAPK hyperactivation in vitro, and treatment of two patients caused rapid improvement in bulk tissue, improving bodily movement and reducing inflammation and severe pruritus. These findings offer a genetic diagnosis to an additional group of patients and trametinib as a treatment option for the severe associated phenotypes.

Topical delivery of seriniquinone for treatment of skin cancer and fungal infections is enabled by a liquid crystalline lamellar phase.

Seriniquinone (SQ) was initially described by our group as an antimelanoma drug candidate and now also as an antifungal drug candidate. Despite its promising in vitro effects, SQ translation has been hindered by poor water-solubility. In this paper, we described the challenging nanoformulation process of SQ, which culminated in the selection of a phosphatidylcholine-based lamellar phase (PLP1). Liposomes and nanostructured lipid carriers were also evaluated but failed to encapsulate the compound. SQ-loaded PLP1 (PLP1-SQ) was characterized for the presence of sedimented or non-dissolved SQ, rheological and thermal behavior, and irritation potential with hen's egg test on the chorioallantoic membrane (HET-CAM). PLP1 influence on transepidermal water loss (TEWL) and skin penetration of SQ was assessed in a porcine ear skin model, while biological activity was evaluated against melanoma cell lines (SK-MEL-28 and SK-MEL-147) and C. albicans SC5314. Despite the presence of few particles of non-dissolved SQ (observed under the microscope 2 days after formulation obtainment), PLP1 tripled SQ retention in viable skin layers compared to SQ solution at 12 h. This effect did not seem to relate to formulation-induced changes on the barrier function, as no increases in TEWL were observed. No sign of vascular toxicity in the HET-CAM model was observed after cutaneous treatment with PLP1. SQ activity was maintained on melanoma cells after 48 h-treatment (IC50 values of 0.59-0.98 µM) whereas the minimum inhibitory concentration (MIC) against C. albicans after 24 h-treatment was 32-fold higher. These results suggest that a safe formulation for SQ topical administration was developed, enabling further in vivo studies.

Active natural compounds perturb the melanoma risk-gene network.

Cutaneous melanoma is an aggressive type of skin cancer with a complex genetic landscape caused by the malignant transformation of melanocytes. This study aimed at providing an in silico network model based on the systematic profiling of the melanoma-associated genes considering germline mutations, somatic mutations, and genome-wide association study signals accounting for a total of 232 unique melanoma risk genes. A protein-protein interaction network was constructed using the melanoma risk genes as seeds and evaluated to describe the functional landscape in which the melanoma genes operate within the cellular milieu. Not only were the majority of the melanoma risk genes able to interact with each other at the protein level within the core of the network, but this showed significant enrichment for genes whose expression is altered in human melanoma specimens. Functional annotation showed the melanoma risk network to be significantly associated with processes related to DNA metabolism and telomeres, DNA damage and repair, cellular ageing, and response to radiation. We further explored whether the melanoma risk network could be used as an in silico tool to predict the efficacy of anti-melanoma phytochemicals, that are considered active molecules with potentially less systemic toxicity than classical cytotoxic drugs. A significant portion of the melanoma risk network showed differential expression when SK-MEL-28 human melanoma cells were exposed to the phytochemicals harmine and berberine chloride. This reinforced our hypothesis that the network modeling approach not only provides an alternative way to identify molecular pathways relevant to disease but it may also represent an alternative screening approach to prioritize potentially active compounds.

Characterization of Immune Cell Populations of Cutaneous Neurofibromas in Neurofibromatosis 1.

Cutaneous neurofibromas (cNFs) are characteristic of neurofibromatosis 1 (NF1), yet their immune microenvironment is incompletely known. A total of 61 cNFs from 10 patients with NF1 were immunolabeled for different types of T cells and macrophages, and the cell densities were correlated with clinical characteristics. Eight cNFs and their overlying skin were analyzed for T cell receptor CDR domain sequences, and mass spectrometry of 15 cNFs and the overlying skin was performed to study immune-related processes. Intratumoral T cells were detected in all cNFs. Tumors from individuals younger than the median age of the study participants (33 years), growing tumors, and tumors smaller than the data set median showed increased T cell density. Most samples displayed intratumoral or peritumoral aggregations of CD3-positive cells. T cell receptor sequencing demonstrated that the skin and cNFs host distinct T cell populations, whereas no dominant cNF-specific T cell clones were detected. Unique T cell clones were fewer in cNFs than in skin, and mass spectrometry suggested lower expression of proteins related to T cell-mediated immunity in cNFs than in skin. CD163-positive cells, suggestive of M2 macrophages, were abundant in cNFs. Human cNFs have substantial T cell and macrophage populations that may be tumor-specific.