Penile Cancer: Innovations in Ultrastructural and Vibrational Markers

Penile Cancer: Innovations in Ultrastructural and Vibrational Markers | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Penile Cancer: Innovations in Ultrastructural and Vibrational Markers Joel Félix Silva Diniz-Filho, Ana Caroline Muniz Silva, Antônio Augusto Lima Teixeira, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4559053/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Penile cancer, although uncommon on a global scale, predominantly arises from squamous cell carcinomas (SCCs). Its occurrence is notably higher in low- and middle-income countries, highlighting the geographic and socioeconomic disparities in the prevalence of this disease. The diversity and significant public health impact of penile cancer underscores the need for new approaches. Nanotechnology, especially through Atomic Force Microscopy (AFM), is promising for studying the nanoscale properties of penile tumor tissues and cells. AFM provides high-resolution topographic images, allowing you to examine the ultrastructural features of cancerous cells and tissues in detail. This helps better understand tumor biomechanics, cell adhesion, morphology, and tumor microenvironment. Raman Spectroscopy (RS) is a powerful technique that detects and analyzes cellular or tissue samples based on morphological characteristics. It scatters photons by molecules polarized by a laser beam, generating a spectral image that reflects the cell's or tissue's chemical composition. This technique can identify changes in the components of cells and tissues, indicative of the presence or progression of the disease. This study proposes to apply RS to investigate the vibrational properties of penile tumor cells and tissues compared with non-tumor counterparts. In this study, through the use of AFM and RS, samples of the subtypes of penile cancer, basaloid and sarcomatoid, as well as non-tumor samples, were analyzed to apply a physical approach to investigate the ultrastructural and vibrational morphology of penile cancer, taking as main tools AFM and RS, providing new information about its nanoscale ultrastructure and offering a new understanding of cancer behavior beyond its molecular composition. Penile Cancer Biomechanics Atomic Force Microscopy Biophysics Raman Spectroscopy Tumor Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION Penile cancer (PCa) presents a significant geographic and socioeconomic disparity in its incidence rates. It is relatively rare in developed regions such as North America and Eastern Europe. In contrast, its prevalence is notably higher in developing countries across Asia, Africa, and South America [ 1 ]. Recent data indicate a concerning uptrend in PCa incidence in these areas, highlighting the disease's impact on public health and underscoring the need for targeted research and healthcare strategies to address this growing challenge [ 2 – 3 ]. Histologically, most penile squamous cell carcinomas (SCCs) share morphological similarities with squamous neoplasms originating in other organs, particularly closely resembling those found in oral and cervical regions. This commonality underscores a shared pathogenesis that might be rooted in squamous cell pathology [ 4 ]. About 70% of penile SCCs are classified as the usual type, characterized by their histological conformity to the classic presentation of squamous cell carcinomas. The remaining 30% of cases are distributed among distinct histological subtypes, including basaloid, verrucous, papillary, and sarcomatoid variants [ 4 – 6 ]. Each of these histological subtypes exhibits unique clinical behaviors and prognostic implications. Within the spectrum of penile squamous cell carcinoma (SCC) subtypes, the basaloid and sarcomatoid variants merit specific focus due to their unique histopathological characteristics and clinical implications. Basaloid SCC, which represents about 10% of penile SCC occurrences, is more aggressive. This subtype is defined by the formation of small cellular clusters exhibiting minimal cytoplasm, pronounced nuclei, and a high frequency of mitotic figures, frequently in necrotic tissue. The designation "basaloid" reflects the morphological similarity of these neoplastic cells to basal cells. Histologically, basaloid SCC is identified by a distinctive architectural feature: a palisade-like configuration of cells at the periphery of tumor islands, coupled with a notable lack of intercellular bridges and the presence of central coagulative necrosis, often referred to as comedonecrosis. These defining characteristics contribute to the diagnostic criteria for basaloid SCC and underscore the subtype's aggressive nature and potential impact on patient prognosis [ 7 ]. Basaloid squamous cell carcinoma (SCC) is characterized by its intimate interaction with the surrounding connective stroma, which supports clusters of basaloid cells. This interaction manifests as distinctive slits or interfaces, commonly referred to as the epithelium-stroma interface. These morphological features facilitate the carcinoma's identification and aggressive behavior and frequently demonstrate a propensity for deep infiltration into the underlying tissues. A significant clinical concern with basaloid SCC is its high rate of lymphatic spread; more than half of the patients exhibit involvement of the inguinal lymph nodes at the time of initial diagnosis. This pattern of aggressive growth and early lymphatic involvement underscores the importance of prompt, accurate diagnosis and comprehensive management strategies to address both the primary tumor and potential metastatic disease [ 8 ]. This subtype of PaC is directly associated with HPV. Human papillomavirus (HPV) represents a pivotal factor in the etiology of penile cancer, a malignancy that, although relatively uncommon globally, poses significant health challenges in various regions, especially in developing countries. HPV, a DNA virus from the Papillomaviridae family, is known for infecting epithelial cells of the skin and mucous membranes, leading to a range of outcomes from benign lesions to malignancies [ 9 , 10 ]. The oncogenic potential of HPV, particularly high-risk subtypes such as HPV 16 and 18, is linked to their genetic material integrating into the host cell's DNA, thereby disrupting normal cell cycle regulation and promoting malignant transformation [ 11 , 12 ]. This process is mediated by the viral oncoproteins E6 and E7, which interfere with tumor suppressor proteins p53 and retinoblastoma (Rb) [ 13 ]. Although rare (occurring in less than 1% of cases), the sarcomatoid subtype demonstrates significant biological aggressiveness, attaining considerable sizes and penetrating deeply into adjacent structures. Microscopically, it is characterized by spindle-shaped cells intermingled with cells of bizarre or giant shapes and may present sarcomatous components, such as chondrosarcoma or osteosarcoma. The histology of sarcomatoid SCC is biphasic, involving the differentiation of squamous epithelial and mesenchymal components. In the transformation process from squamous cells to spindle cells, the epitheliomesenchymal transition occurs through a decrease in the expression of E-cadherin, the primary epithelial intercellular adhesion molecule, and an increase in the expression of N-cadherin, responsible for the mobile phenotype of the cells [ 14 ]. The limited evidence on penile squamous cell carcinoma with epithelioid features (PCa) underscores a critical gap in our comprehensive understanding of its tumor biology. To bridge this knowledge gap, this study integrates Atomic Force Microscopy (AFM) and Raman Vibrational Spectroscopy (RS) as investigative tools for a detailed ultrastructural and molecular examination of PCa tumors, specifically focusing on sarcomatoid and basaloid subtypes. The juxtaposition of ultrastructural and molecular data from these advanced imaging techniques promises to shed light on the complex biology of penile squamous cell carcinoma, potentially paving the way for new approaches to its unique pathological features. METHODOLOGY Tissue Selection Specialized researchers collected the samples from three reference hospitals located in São Luís, Maranhão (Presidente Dutra University Hospital, Aldenora Bello Cancer Hospital, and Maranhão Cancer Hospital Dr. Tarquínio Lopes Filho). Participants were informed about the study's research objectives, risks, and expected impacts. All participants signed the Informed Consent Form (ICF), which the Human Research Ethics Committee approved. Subjects who agreed to participate in the research were interviewed for socio-behavioral data collection using a data collection instrument. In contrast, those who did not agree were assured that there would be no harm to conventional hospital treatment and follow-up. After the material was collected for research, all materials were identified using a specific project code to ensure participants' confidentiality and privacy rights. Inclusion Criteria This study considered men over 18 years of age with clinical and anatomopathological diagnoses of penile cancer who had an amputation as the first therapeutic option. Only those who agreed to participate in the study by signing the Informed Consent Form (ICF) were included. Exclusion Criteria Those who had undergone chemotherapy or radiotherapy before the surgical procedure were excluded. Review of Histological Slides and Selection of Study Area After applying inclusion and exclusion criteria, cases were selected, and their Hematoxylin and Eosin (H&E) stained slides underwent a reevaluation to confirm histological diagnosis and tumor classification according to the criteria proposed by the American Joint Committee on Cancer (AJCC) [ 15 ]. The slide review was conducted independently by two pathologists. Tumor subclassification (CECs) was based on criteria established in the medical literature [ 16 ]. Tissue Collection The samples used in this study were collected in the surgical center: the physician responsible for the amputation collected small fragments of fresh tissue containing tumor and non-tumor (normal) samples. The samples were stored in the following solutions: (1) RNAlater (ThermoFisher TM) for DNA extraction and HPV detection and (2) 10% buffered formalin for biophysical analyses. HPV Detection and Genotyping The QIAamp Fast DNA Tissue kit (Qiagen, Cat. No. 51404) was used for DNA extraction. The extracted samples were evaluated for extraction quality by quantifying the total material on a NanoDrop spectrophotometer (ThermoFisher TM), with concentrations expressed in ng/µL, and purity assessment with 260/280 nm measurements (between 1.8 and 2.0) and 260/230 (above one). The samples were stored at -20°C until used in subsequent steps. HPV detection was conducted by conventional PCR (Polymerase Chain Reaction) in two stages (nested PCR). In the first PCR, a set of generic primers called PGMY09/11, described by Gravitt et al. [ 17 ], which produced a 450 bp fragment of the HPV capsid L1 region, was used. The primer GP5+/6 + was used in the second PCR, generating a 170 bp amplicon corresponding to the viral capsid L1 region. A pair of primers for the β-globin gene (366 bp fragment) was used as a positive control for the reaction. The final mix was 25 µL for each sample, using the MASTERMIX PCR PLATINUM SUPERFI kit (Life TechnologiesTM), followed by 45 cycles of 94°C for 45 seconds, 40°C for 1 minute, and 72°C for 1 minute; and finally 72°C for 10 minutes. The amplicons were separated on a 1.5% agarose gel and subjected to a constant voltage of 90V for 40 minutes. Only those with amplification for the β-globin and GP5+/6 + genes were considered positive. Capillary electrophoresis sequenced positive cases, and their sequences were compared to those available in genetic databases using BLAST (Basic Local Alignment Search Tool) for viral genotyping. Tissue Preparation The PCa samples will be embedded in paraffin and cut using an ultramicrotome (model LEICA EM UC6), producing 2 µm thick sections. The biopsies will be deposited on 13 mm diameter glass slides and subsequently placed in an oven at 60 degrees for 30 minutes for dewaxing. After this process, the samples will be submerged in 30 ml of Xylene and gently shaken for 15 minutes, with 30 ml of Xylene being changed every 5 minutes. Subsequently, the samples will be rehydrated through a sequence of 90%, 80%, and 70% PA ethyl alcohol for tissue rehydration. Atomic Force Microscopy Setup The analysis by Atomic Force Microscopy was conducted using an AFM Multimode 8 (Bruker, Santa Barbara, CA, USA) in PeakForce Quantitative Nanomechanics mode - QNM. For this purpose, probes of the qp-HBC model (NanoSensors) with a nominal cantilever spring constant of 0.5 N/m and a tip radius smaller than 10 nm were utilized. All data were obtained with a scanning rate of 0.5 Hz and a curve acquisition frequency of 0.5 kHz. Three non-tumorous samples (Control Group), three samples of the sarcomatoid subtype, and three samples of the basaloid subtype were used. Each sample underwent 15 scans at distinct points. In total, 45 maps of 25µm x 25µm were analyzed for each group. Each scan contained 65536 force curves, providing a broad database for comparative analysis among the studied groups. Ultrastructural Analysis For AFM data, the statistical roughness analysis was based on the height of each pixel in the image, analyzed from the height map, according to the methodology described by Rates et al. [ 18 ]. Area and volume data were calculated from topographic maps of 25 x 25 µm of tissue surface (control/tumor) using the Gwyddion 2.57 software. The deformation parameter was calculated using the ratio \(A/V\) . According to Degiorgio et al. [ 19 ], changes in surface area and volume are related to changes in cell (tissue) membrane composition and membrane (tissue) component arrangements about changes in the cell´s surface area and volume and, consequently, in cell (tissue) deformability. Hole diameters for basaloid (n = 669) and sarcomatoid tissue (n = 94) were calculated using Gwyddion 2.57 software, applying the threshold edge detection data tool. Values were expressed as mean ± SD. Raman Spectroscopy Analysis Raman Spectroscopy was used to analyze and identify the spectral differences obtained from the control group and groups of patients with PCa, previously disclosed through the clinical method. The measurements were carried out on Horiba’s T64000 spectrometer with a CCD (Charge Coupled Device) detection system cooled with liquid nitrogen. All measurements were obtained in backscatter geometry. The 532 nm line was used as a traction source with its maximum power for the measurements. The sample surface was viewed using a specific Olympus brand with an attached video camera. To focus the brightness on the surface, we used a 100 \(\times\) lens. Nine acquisitions were carried out with times of 20 seconds. The spectral region observed in our experiments was divided into intervals of 750 to 1750 \(c{m}^{-1}\) (Low Wavenumbers - LWN) and 2650 to 3150 \(c{m}^{-1}\) (High Wavenumbers - HWN) Spectral Pre-Processing Data processing was conducted using LabSpec6 software. The narrow peaks caused by cosmic rays were sequentially removed, and the variable fluorescence background and the glass substrate were estimated using a fifth-order polynomial fitting and subsequently subtracted. Each spectrum was smoothed using a polynomial smoothing algorithm before analysis. Principal Component Analysis - PCA Principal Component Analysis (PCA) was applied to the spectral dataset, a statistical analysis method capable of reducing the dimensionality of the data while capturing most of the variation in the original dataset. The spectra were analyzed following the methodology of Yi Hong Ong and coworkers [ 20 ], where variance analysis was employed on the scores of the first ten principal components to determine which PC exhibited significant differences in mean scores between the two groups of cells, utilizing OriginLab software. Statistical Analysis Statistical test following a single criterion was evaluated using ANOVA and Tukey’s post-test, considering the values were statistically significant when p < 0.05. Statistical analyses and graphics were performed using the ORIGIN software. The calculated error was the standard deviation (SD) in all data. RESULTS Figure 1 shows a representative optical microscopy image of each tissue analyzed. In Fig. 1 A, representing the control group, the histological slide shows the layers of the epidermis with stratified squamous cells, without neoplastic changes, with the stratum corneum being the outermost layer with a pink color, as shown by the arrow, and the basal layer being the innermost with large, elongated and hyperchromatic nuclei pigmented with hematoxylin [ 21 ]. Figure 1 B shows the basaloid group, indicated by the abnormal growth of bluish, small, uniform cells with round nuclei and scant cytoplasm, which resemble the basal cells of epithelial tissue, normally presenting a palisade arrangement in the peripheral cells of the tumor islets, absence of intercellular bridges and the presence of central coagulation necrosis [ 22 – 23 ]. Figure 1 C represents the sarcomatoid group, marked by the differentiation of squamous and mesenchymal components, characterized by the expression of spindle-shaped sarcomatous cells, which exhibit atypical and elongated nuclei, resulting from the epitheliomesenchymal transition, in which the squamous cell is transformed into spindle cells [ 24 ]. Such histopathological components show the conformational changes undergone by the pathological tissue compared to the control group. The representative high-resolution AFM maps of each group reveal ultrastructural changes on the surface of PCa tissues, as observed in Fig. 2 . Figure 2 A shows a 25 µm 2 scan over a non-tumor region of penile tissue and its respective 3D view in Fig. 2 D, compatible with the preserved ultrastructural morphology of the stratum spinosum layer [ 25 ]. In contrast, Fig. 2 B (Fig. 2 E 3D view) shows a scan of the same size for basaloid cancer tumor tissue associated with HPV infection [ 26 ]. The submicrometric-sized holes in Fig. 2 B may be related to the percolation or diffusion of viral particles in this type of tumor. In PCa sarcomatoid subtype tissue, as shown in Fig. 2 C (Fig. 2 F 3D view), several stretches of tissue form micrometric holes on its surface. This fact is associated with high vascularity in cancer tissues, indicating rapid tumor growth, as vascularization is necessary to supply nutrients and oxygen to cancer cells [ 27 ]. In Fig. 3 , one can see how each PCa subtype promotes fenestrations in the tumor tissue. Figure 3 A shows a representative image of the basaloid subtype, showing uniform fenestrations (or pores) of submicron diameter (0.688 ± 0.053 µm) (mean ± SD). As with many medications [ 28 – 31 ], which prevent or reduce its effectiveness, viral particles can become trapped by this complex porous structure, which may associate this subtype with HPV infection. Figure 3 B shows a representative image of the surface detail of the sarcomatoid subtype tumor tissue. Here, it is possible to observe a greater presence of dark regions (holes), micrometric in size (6.39 ± 1.28 µm), compatible with tissue failures. These holes have a medium diameter compatible with capillaries that irrigate the tumor tissue [ 32 , 33 ]. When the vascular and nutritional supply aligned with the high mitotic activity of the tumors does not supply the demands of the tumor microenvironment, the formation of foci of necrosis, typical in basaloid and sarcomatoid PCa, occurs, visible through the stretches [ 34 ]. Furthermore, high vascularity can also facilitate the spread of cancer to other parts of the body through the bloodstream. This stretching may also be linked to the cytoskeleton and extracellular matrix collagen’s structural changes that gradually promote cancer progression [ 35 – 37 ]. Motivated by these qualitative ultrastructural differences observed in tumor tissues concerning non-tumor tissue and also between the different types of tumors (basaloid and sarcomatoid), we analyzed quantitative ultrastructural parameters of the groups, such as mean quadratic roughness of the tissue surface, surface area, tissue image volume and deformation (A/V ratio). The results can be seen in the panel shown in Fig. 4 . The scatterplot shown in Fig. 4 A presents the mean squared roughness results for each group analyzed. The mean values ​​and their respective standard deviations are 283.8 ± 8.2 nm, 360.9 ± 12.3 nm, and 284.1 ± 8.1 nm for the control, basaloid, and sarcomatoid tissues. A greater number of holes (fenestrations) in the basaloid tumor tissue is reflected in the increased roughness result, which may be associated with the greater capacity of these tumors to trap a greater quantity of viral particles [ 38 ]. Figure 4 B shows the scatter plot of surface area values ​​of tissues from each group analyzed. The average values ​​obtained were 810.4 ± 10.1 µm 2 , 738.2 ± 4.6 µm 2 , and 767.4 ± 5.7 µm 2 , respectively for the control, basaloid and sarcomatoid groups. Here, it is possible to observe a trend, with statistical relevance, of a decrease in the surface area of ​​tumor tissues when compared with non-tumor ones. Figure 4 C presents the volume results of the maps obtained from the analyzed tissues. The average values ​​obtained were 940.8 ± 38.0, 656.4 ± 29.4, and 395.2 ± 18.5 µm 3 for the control, basaloid and sarcomatoid groups, respectively. As with the surface area, the maps obtained from the tumor tissue samples showed a reduced average volume compared to the control group. The graph shown in Fig. 4 D shows the scatter plot of surface deformation calculated from the geometric parameters of the images obtained from each group. The average values ​​found were, respectively, 0.91 ± 0.03, 1.2 ± 0.04, and 2.1 ± 0.07 µm − 1 for the control, basaloid and sarcomatoid groups. It is possible to observe greater deformability in tumor samples than in non-tumor ones. This fact may be associated with the greater capacity for tumor deformation at the cellular level [ 39 , 40 ] since we are analyzing the ultrastructure of tumor tissues, which enables greater invasion of these tumors into new sites. Another promising approach is investigating the vibrational signature obtained through Raman Spectroscopy in the tissues examined. This biochemical study, combined with AFM data, may bring new perspectives to the study of penile tumors. Figure 5 shows the average spectrum obtained from 30 samples from each group. The bands refer to the vibrational modes associated with the main biochemical components of tissues, such as proteins and amino acids, carbohydrates, and lipids, among others, for both low and high wavelengths. This way, it is possible to observe the differences in the biochemical composition among the tissues analyzed. The specific wavelengths for each identified mode can be found in Table 1. Table 1: Assignments of each mode of the tissue Raman spectrum [41– 46]. Wavenumber (cm -1 ) Amino acid/ Protein Lipid/ Carbohydrate Other 813 C – C str. 856 Proline 874 C – C str. 888 Protein 919 Proline 937 Proline 1002 Phenylalanine 1031 Phenylalanine 1058 Lipids 1100 Lipids Fatty acid 1131 Phospholipids 1159 C – C / C – N str. 1169 Proline 1207 Hydroxyproline, Tyrosine 1239 – 1272 Amide III 1293 Cytosine 1315 Guanine 1340 Nucleic Acid 1381 CH 3 1392 C – N str. 1402 Methyl ben groups. 1416 C = C str. 1450 CH 2 ben. 1458 Nucleic Acid 1514 Cytosine 1638 – 1665 Amide I 2728 C – H str. 2853 CH 2 sym. str. 2888 CH 2 asym. str. 2935 CH 3 sym. str. CH 3 sym. str. 2960 – 2980 CH 3 asym. str. CH 3 asym. str. 3008 = CH str. 3030 Aromatic Aromatic Abbreviation: str. = stretching, sym. = symmetric, asym. = asymmetric, def. = deformation, ben. = bending. To evaluate the discriminatory capacity of the method used through multivariate analysis, Principal Component Analysis (PCA) was performed on all data contained in the Low Wavenumber (LWN) and High Wavenumber (HWN) regions). In total, 30 spectra from each sample group were analyzed, suitable for statistical analysis. The ellipses present in the graph delimit the area in which 95% of the data is included. In Figure 6, the first three main components are highlighted, which result in good total variability of the data set. Figure 6A shows the confidence ellipses for the control and sarcomatoid groups considering the spectrum region between 750-1750 cm -1 (low wavelengths - LWL). The first three main components add up to 81.9% confidence. Figure 6B shows the relationship between the same groups, now for high wavelengths (HWL), between 2650-3150 cm -1 . In this spectrum range, the first three PCs total 97.2% confidence. Figure 5C shows the confidence ellipses for the control and basaloid groups for LWL, with a confidence of 82.3% for the first three PCs. In contrast, Figure 6D shows the relationship between the same groups for HWL, with a confidence of 95.4 % for the first three PCs. Figures 6E and F show the relationship between the tumor groups (basaloid and sarcomatoid) for LWL (76.2%) and HWL (96.3%), respectively. DISCUSSION The results suggest that cytoskeleton-induced modifications can change tumor tissue morphology. Sarcomatoid PCa has a biphasic character, marked by a squamous component with sarcomatous differentiation of spindle cells [14]. This epitheliomesenchymal transition is characterized by decreased expression of E-cadherin and increased expression of N-cadherin, which is responsible for the mobile phenotype of the cells [14]. Cadherins are polypeptides responsible for epithelial intercellular adhesion, associated with a group of catenin proteins that bind the actinic microfilaments of the cytoskeleton [47]. Zemła and coworkers demonstrated that the most rigid conformations within the cell surface are made up of actin filaments, and the structural disarrangements in the organization of the cytoskeleton were attributed to lower cellular rigidity, giving a mobile aspect to cancer, which correlates with the modulations caused by losses of E-actin-bound cadherins [48]. Changes in cytoskeletal dynamics, mediated by changes in cadherin expression, can influence cell morphology, indicated by changes in area and volume, which express a reduction in volume and area data in the sarcomatoid group. Another possible explanation to describe the structural and functional changes of the actin cytoskeleton is L-plastin, a group of actin-bridging proteins that contribute to tumor cell invasion in a phosphorylation-dependent manner [35]. Phosphorylation of L-plastin at its Ser5 residue increases its ability to interact with actin, thus influencing its intracellular localization [35]. The supply of energy to trigger phosphorylation on the L-plastin residue may be associated with glycolytic enzymes, which in cancer cells, due to the high rate of glycolysis, are increased, producing ATP in the vicinity of the cytoskeleton through reversible binding of glycolytic enzymes to the cytoskeleton [51]. PCa has subtypes associated with HPV, such as basaloid, and non-associated subtypes, such as sarcomatoid [2]. HPV-related penile carcinogenesis, typical of the basaloid subtype, arises from the overexpression of the viral oncoproteins E6 and E7, causing cell cycle dysregulation and genomic instability [38]. The viral oncoprotein E6 interferes with the p53 pathway, a tumor suppressor protein, inhibiting apoptosis by targeting the protein for degradation. The inhibition of p53 by E6 promotes exacerbated cell proliferation and tumor cell immortalization. However, non-HPV-associated carcinogenesis, such as in the sarcomatoid subtype, may result from mutagenic changes in tumor suppressor genes [49]. In a study carried out by Jacob et al., patients with metastatic penile cancer had mutations in TP53 [50]. They were negative for HPV, suggesting that mutations in TP53 and consequent overexpression of p53 were associated with metastasis in patients with advanced cancer and decreased patient survival [2]. The P53 pathway is a regulator in the formation of tumor-associated collagen signature 3, a collagen bundle angled at 60° to 90° to the edge of the cancer and is indicated by cancer proliferation and invasion [36]. The changes show that the sarcomatoid subtype presents a specificity in the expression of collagen in the extracellular matrix that can alter its nanomechanical and ultrastructural properties, such as the reduction in area and volume, associated with ECM modulations that favor the formation of apertures. The modification in surface roughness may be associated with biological processes underlying cancer development, such as uncontrolled cell proliferation and reorganization of the ECM [51]. Metastatic cancer cells exhibit an expanded expression of transport proteins such as ion channels, ion transporters, and aquaporins. These ion/water transport proteins, such as NHE1, NKCC1, AE2, ENaC, AQPs, IK channel, VRACs, ClC-3, and TMEM16s, often demonstrate elevated activity or expression in cancer cells. The increase in expression of these membrane proteins may justify the roughness observed in basaloid and sarcomatoid PCa, indicating a possible adaptation of cancerous tissues for more effective and invasive dissemination in other tissues [52]. There was a reduction in the surface area of PCa tissues, which was more evident in the basaloid subtype, affected by the HPV virus. When comparing the samples with the control group, a similar pattern can be seen in the maps, with a decrease in height between the sarcomatoid and basaloid samples. This reduction in surface area suggests greater aggressiveness of the tumor, especially in the basaloid and sarcomatoid subtypes, classified as aggressive and with a high rate of nodal metastasis [53,54]. Compared to control samples, the analysis of basaloid and sarcomatoid samples revealed a reduction in volume. Transport proteins, such as ion channels, ion transporters, and aquaporins (AQPs), regulate cell volume during exposure to osmotic stress [55]. Studies indicate that water flow related to osmotic gradients generated by ionic transport contributes to cell migration [56 – 59]. It was reported that cell migration is attenuated by extracellular hypertonicity; cell shrinkage, which inhibits local volume, would facilitate cell migration [60]. Furthermore, the osmotic gradient is responsible for regulating the expression of ion/water transport proteins and their changes in location in the membrane, modulating the cycles of protrusion of the leading edge and retraction of the rear part of the cell during migration [61]. These aspects demonstrate that changes in protein expression in metastatic cells, altered by extracellular osmotic stress, directly impact cell migration, typical of metastatic cancer. The correlation with volume and deformation data shows that changes in cellular structure caused by the osmolarity of the medium can impact the reduction of metastatic cell volume and increase cellular deformation of the basaloid and sarcomatoid subtypes compared to the control group. The rapid growth of cancer cells can exceed blood supply capacity, leading to areas of necrosis due to a lack of oxygen and nutrients [62,63]. Compression of surrounding blood vessels can result in atrophy [64]. Both cancer subtypes, basaloid and sarcomatoid, show high rates of mitosis and areas of necrosis, with basaloid characterized by comedonecrosis. Excessive mitotic activity concerning vascular and nutritional supply can result in tissue necrosis [65, 66], influencing the area and volume measurements observed in AFM. Hypoxic conditions in the collagen-rich ECM, intensified by the interaction between cancer cells and collagen, affect vascular supply. Factors such as HIF-1, LOX, and metalloproteinase play roles in this process, as they are related to cancerous blood vessels. The firmness of collagen in the matrix affects vascular growth, impacting the formation of necrotic foci and fissures identified in AFM [50]. The increase in deformation in the basaloid and sarcomatoid groups can be explained by the same mechanisms, given that the deformation is directly correlated with area and volume. Raman spectroscopy analysis strongly corroborates these data. When a molecular group changes, the vibrational modes relative to it are also changed. Changes in the intensity, position, and broadening of the Raman spectrum peak can verify this fact. The greatest discrepancies between control penis tissues and tissues appear in modes located in the low wavenumber range (LWN) 700 to 1800 cm -1 , where vibrational modes related to proteins are observed, such as the of Proline (919 cm -1 ), amino acids are essential amino acids that are abundant in structural proteins like collagen, and stretching of the C – H and C = C bonds (1392 cm -1 and 1416 cm -1 respectively). In the range between 1239 and 1272 cm -1 ), attributed to Amide III and in the region 1638 to 1665 cm -1 , attributed to Amide I, which are groups composed of carbon, oxygen, and nitrogen atoms (CONH), plays a crucial role in the formation of proteins. These bonds are essential for conferring structural rigidity and provide information about secondary structure organization in PCa tissues. Furthermore, changes were identified in the bands corresponding to lipids (1131 and 1381 cm -1 ). These modes reflect the composition, organization, and structure of lipids in penile cancer tissues, providing valuable information about the tissue’s biochemistry. The presence of cytosine (1514 cm -1 ) in PCa tissues indicates how mutations or epigenetic changes can be critical in transforming a normal cell into a cancerous cell [67 – 68]. In the high wavenumber region (HWN) of the Raman spectrum, between 2700 and 3100 cm -1 ), stretch bands of the C – H bonds of lipids present in the membranes of PCa tissues are detected, described by Matthews et al. [69]. Despite not demonstrating the precursor of lipid breakdown, this result shows that these components are most likely related to carcinogenic transformation. These vibrations also provide crucial information about the composition and organization of lipids in the lipid layers of PCa tissues, playing a fundamental role in membrane integrity and permeability. In the same way, as in the low wavelength region, we also observed variations in the intensities of the modes related to the biochemical groups associated with PCa. According to the principal component analysis (PCA) of the spectra, a statistically significant distinction can be observed between the spectra obtained in the control and sarcomatoid and basaloid samples. This statistically substantial distinction indicates differences in the spectral characteristics of these groups. These differences can be explored to develop more accurate and efficient assessment methods for these types of tissues. When performing an analysis of the spectral data of the basaloid and sarcomatoid subtypes, statistical significance was found, indicating the differences in the spectral characteristics. The fact that the ellipses are close together suggests a relationship or similarity between the basaloid and sarcomatoid subtypes. This fact means that although there are statistically significant differences in spectral characteristics, overlap or proximity exists in some areas of these subtypes. These results are important because they suggest exploring the similarities and differences identified to develop more accurate and efficient assessment methods to distinguish between control, basaloid, and sarcomatoid groups. In other words, multivariate analysis provides information about the global variation in spectral data. It highlights different and shared aspects between these groups, which can be useful in developing more refined assessment approaches. CONCLUSION The study investigated the ultrastructural and vibrational properties of tissue subtypes of penile cancer (PCa) using Atomic Force Microscope (AFM) and Raman Spectroscopy (ER). AFM maps revealed ultrastructural changes in PCa tissues, indicating wear and stretching, possibly due to cytoskeleton and ECM reorganization. Increased roughness of tissues affected by cancer suggests membrane erosion related to tumor invasion. The loss of proteins and lipids in cancer cells may contribute to this roughness, affecting cellular communication and cancer growth and the increased expression of transport proteins, such as ion channels, ion transporters, and aquaporins in metastatic cancer cells. Analysis of the surface area showed a reduction, especially in the basaloid subtype, associated with aggressiveness and high nodal metastasis. The decrease in basaloid and sarcomatoid sample volumes suggests possible necrosis, lack of blood supply, and cell volume regulation during exposure to osmotic stress. High rates of mitosis and areas of necrosis in these subtypes influence area and volume measurements. Hypoxic conditions in the collagen-rich extracellular matrix, related to factors such as HIF-1, LOX, and Metalloproteinase, affect vascular supply. ER detected vibrational modes in PCa tissues, revealing spectral differences between control and cancerous samples. Multivariate analysis confirmed the techniques' discriminatory capacity, indicating significant differences between control and sarcomatoid/basaloid samples. These discoveries could potentially promote precise and efficient physical approaches to cancer, allowing for earlier detection and more targeted treatment. Understanding these characteristics represents a significant advance in oncology. Declarations ACKNOWLEDGMENTS : The authors thank CAPES (001), UFMA, HUUFMA, and FAPEMA for supporting this research development. FUNDING : This study was funded by CAPES Financial Code 001, FAPEMA, Projeto Universal-06929/22, Bolsa de Produtividade CNPq, Luciana Magalhães Rebelo Alencar - 304774/2021-9. CONFLICTS OF INTEREST : All authors declare no conflict of interest. ETHICS APPROVAL AND CONSENT TO PARTICIPATE: The study received approval from the Research Ethics Committee on Human Subjects at the University Hospital of the Federal University of Maranhão (CEP/HU-UFMA) (CAAE 30760420.3.0000.5086) and was conducted by the Declaration of Helsinki. Considering the study's retrospective nature, the Research Ethics Committee on Human Subjects at the University Hospital of the Federal University of Maranhão exempted the need for patient consent (Opinion Number 4.228.789). References Christodoulidou M, Sahdev V, Houssein S, Muneer A (2015) Epidemiology of Penile Cancer. Curr Probl Cancer 39:126–136 Teixeira Júnior AAL, da Costa Melo SP, Pinho JD, Sobrinho TBM, Rocha TMS, Duarte DRD, de Oliveira Barbosa L, Duarte WE, de Castro Belfort MR, Duarte KG, da Silva Neto AL, de Calixto RR, Paiva Paiva J, do Nascimento LC, Alencar Junior FSMS, Khayat AM, Lages AS, dos Reis JS, Araújo RB, Silva WS (2022) G.E.B. A Comprehensive Analysis of Penile Cancer in the Region with the Highest Worldwide Incidence Reveals New Insights into the Disease. BMC Cancer , 22 , 1063 Coelho RWP, Pinho JD, Moreno JS, Garbis DVEO; do, Nascimento AMT, Larges JS, Calixto JRR, Ramalho LNZ, da Silva AAM, Nogueira LR, de Moura Feitoza L, Silva (2018) G.E.B. Penile Cancer in Maranhão, Northeast Brazil: The Highest Incidence Globally? BMC Urol , 18 , 50 Sanchez DF et al (2015) HPV- and non-HPV-related subtypes of penile squamous cell carcinoma (SCC): Morphological features and differential diagnosis according to the new WHO classification (2015)., Semin. Diagn. Pathol. , vol. 32, n o 3, pp. 198–221, maio Cubilla AL et al (2018) The World Health Organisation 2016 classification of penile carcinomas: a review and update from the International Society of Urological Pathology expert-driven recommendations, Histopathology , vol. 72, n o 6, pp. 893–904 Cubilla AL, Reuter V, Velazquez E, Piris A, Saito S, Young eRH (2001) Histologic classification of penile carcinoma and its relation to outcome in 61 patients with primary resection., Int. J. Surg. Pathol. , vol. 9, n o 2, pp. 111–120, abr Henry MAC, de Lerco A, Oliveira MM, de Rodrigues WK (2007) Carcinoma basalóide escamoso: uma forma rara e agressiva de câncer do esôfago e revisão da literatura. ABCD arq bras cir dig 20:62–64 Cubilla AL (2009) The Role of Pathologic Prognostic Factors in Squamous Cell Carcinoma of the Penis. World J Urol 27:169–177 Organization WH, INTERNATIONAL AGENCY FOR RESEARCH ON CANCER IARC Monographs on the (2007) Evaluation of Carcinogenic Risks to Humans: Volume 90 Human Papillomaviruses, vol. 90 Gheit T (2019) Mucosal and Cutaneous Human Papillomavirus Infections and Cancer Biology. Front Oncol 9:355 Williams VM, Filippova M, Soto U, Duerksen-Hughes ePJ (2011) HPV-DNA integration and carcinogenesis: putative roles for inflammation and oxidative stress., Future Virol. , vol. 6, n o 1, pp. 45–57, jan Da MLR, Silva et al (2021) jul., The role of HPV-induced epigenetic changes in cervical carcinogenesis (Review)., Biomed. reports , vol. 15, n o 1, p. 60 Yim E-K, Park eJ-S (2005) The role of HPV E6 and E7 oncoproteins in HPV-associated cervical carcinogenesis., Cancer Res. Treat. , vol. 37, n o 6, pp. 319–324, dez Silva DFB, Santos HB, de León P, Gomes JE, de Alves DQ, Nonaka PM (2019) Clinicopathological and Immunohistochemical Analysis of Spindle Cell Squamous Cell Carcinoma of the Tongue: A Rare Case. Einstein (Sao Paulo) 17:eRC4610 WHO.Tumours of the penis (2016) In: Who Classification of Tumours of the urinary system and male genital organs. Vol. 4, pp. 259–285 Edge SB, Byrd DR, Compton CC, Fritz AG, Greene FL (2009) Trotti. AJCC Cancer Staging Manual, 7th edn. Springer, New York, pp 1–649 Gravitt PE et al (jan. 2000) Improved amplification of genital human papillomaviruses. J Clin Microbiol 38:357–361n o 1 Dias Rates ER, Almeida CD, de Costa PF, Jansen E, de Farias M, Santos-Oliveira R, Rebelo R, Alencar LM (2024) Evaluation of Biophysical Alterations in the Epithelial and Endothelial Layer of Patients with Bullous Keratopathy. Exp Eye Res 240:109791 Degiorgio V, Corti M (1985) Physics of Amphiphiles: Micelles, Vesicles and Microemulsions: Proceedings of the International School of Physics, Enrico Fermi, Course Xc ; Degiorgio, V.; Corti, M., Eds.; 1st ed.; ‎ Elsevier Science Ltd, ; Vol. 1 Ong YH, Lim M, Liu Q (2012) Comparison of Principal Component Analysis and Biochemical Component Analysis in Raman Spectroscopy for the Discrimination of Apoptosis and Necrosis in K562 Leukemia Cells. Opt Express OE 20:22158–22171 Prado-Mantilla A, Lechler T (2023) Polarity in skin development and cancer. Curr Top Dev Biol 154:317–336 Baněčková M, Cox D (2023) Top 10 Basaloid Neoplasms of the Sinonasal Tract. Head Neck Pathol 17(1):16–32 Goyal S, Rathore R, Sharma S, Arora VK, Das GK, Singal A (2017) Cutaneous basal cell carcinoma with mixed histology: Cytomorphological features of two unusual cases. J Cytol 34(2):115–118 Raza MA, Mazzara PF (2011) Sarcomatoid carcinoma of esophagus. Arch Pathol Lab Med 135(7):945–948 Losquadro WD (2017) Anatomy of the Skin and the Pathogenesis of Nonmelanoma Skin Cancer, Facial Plast. Surg. Clin. North Am. , vol. 25, n o 3, pp. 283–289 Humphrey PA, Moch H, Cubilla AL, Ulbright TM, Reuter eVE (jul. 2016) WHO Classification of Tumours of the Urinary System and Male Genital Organs-Part B: Prostate and Bladder Tumours. Eur Urol 70:106–119n o 1 Ribatti D, Pezzella eF (2021) Overview on the Different Patterns of Tumor Vascularization., Cells , vol. 10, n o 3, mar Huang H-S et al (2012) Novel antivirals inhibit early steps in HPV infection., Antiviral Res. , vol. 93, n o 2, pp. 280–287, fev Habiger C, Jäger G, Walter M, Iftner T, Stubenrauch eF (2016) Interferon Kappa Inhibits Human Papillomavirus 31 Transcription by Inducing Sp100 Proteins., J. Virol. , vol. 90, n o 2, pp. 694–704, jan Yan H et al (2019) Efficient Inhibition of Human Papillomavirus Infection by L2 Minor Capsid-Derived Lipopeptide., MBio , vol. 10, n o 4, ago Michalczyk K, Misiek M, Chudecka-Głaz eA (2022) Can Adjuvant HPV Vaccination Be Helpful in the Prevention of Persistent/Recurrent Cervical Dysplasia after Surgical Treatment?-A Literature Review., Cancers (Basel). , vol. 14, n o 18, set Takeishi N, Imai eY (2017) Capture of microparticles by bolus flow of red blood cells in capillaries., Sci. Rep. , vol. 7, n o 1, p. 5381, jul Murrant e CL, Fletcher NM (2022) Capillary communication: the role of capillaries in sensing the tissue environment, coordinating the microvascular, and controlling blood flow., Am. J. Physiol. Heart Circ. Physiol. , vol. 323, n o 5, pp. H1019–H1036, nov Velazquez EF, Melamed J, Barreto JE, Aguero F, Cubilla AL (2005) Sarcomatoid Carcinoma of the Penis: A Clinicopathologic Study of 15 Cases. Am J Surg Pathol 29:1152–1158 Al Tanoury Z, Schaffner-Reckinger E, Halavatyi A, Hoffmann C, Moes M, Hadzic E, Catillon M, Yatskou M, Friederich E (2010) Quantitative Kinetic Study of the Actin-Bundling Protein L-Plastin and of Its Impact on Actin Turn-Over. PLoS ONE 5:e9210 Xu S, Xu H, Wang W, Li S, Li H, Li T, Zhang W, Yu X, Liu L (2019) The Role of Collagen in Cancer: From Bench to Bedside. J Translational Med 17:309 Meira DD, Marinho-Carvalho MM, Teixeira CA, Veiga VF, Da Poian AT, Holandino C, de Freitas MS, Sola-Penna M (2005) Clotrimazole Decreases Human Breast Cancer Cells Viability through Alterations in Cytoskeleton-Associated Glycolytic Enzymes. Mol Genet Metab 84:354–362 Yim E-K, Park J-S (2005) The Role of HPV E6 and E7 Oncoproteins in HPV-Associated Cervical Carcinogenesis. Cancer Res Treat 37:319–324 Chen J, Weihs D, Van Dijk M, Vermolen eFJ (2018) A phenomenological model for cell and nucleus deformation during cancer metastasis., Biomech. Model. Mechanobiol. , vol. 17, n o 5, pp. 1429–1450, out Yu W et al (2022) Cancer cell mechanobiology: a new frontier for cancer research, J. Natl. Cancer Cent. , vol. 2, n o 1, pp. 10–17 Madzharova F, Heiner Z, Gühlke M, Kneipp J (2016) Surface-Enhanced Hyper-Raman Spectra of Adenine, Guanine, Cytosine, Thymine, and Uracil. J Phys Chem C 120:15415–15423 Movasaghi Z, Rehman S, Rehman IU (2007) Raman Spectroscopy of Biological Tissues. Appl Spectrosc Rev 42:493–541 Rygula A, Majzner K, Marzec KM, Kaczor A, Pilarczyk M, Baranska M (2013) Raman Spectroscopy of Proteins: A Review. J Raman Spectrosc 44:1061–1076 Czamara K, Majzner K, Pacia MZ, Kochan K, Kaczor A, Baranska M (2015) Raman Spectroscopy of Lipids: A Review. J Raman Spectrosc 46:4–20 Pezzotti G (2021) Raman Spectroscopy in Cell Biology and Microbiology. J Raman Spectrosc 52:2348–2443 Dodo K, Fujita K, Sodeoka M (2022) Raman Spectroscopy for Chemical Biology Research. J Am Chem Soc Ozawa M, Kemler R (1998) Altered Cell Adhesion Activity by Pervanadate Due to the Dissociation of Alpha-Catenin from the E-Cadherin.Catenin Complex. J Biol Chem 273:6166–6170 Zemła J, Danilkiewicz J, Orzechowska B, Pabijan J, Seweryn S, Lekka M (2018) Atomic Force Microscopy as a Tool for Assessing the Cellular Elasticity and Adhesiveness to Identify Cancer Cells and Tissues. Semin Cell Dev Biol 73:115–124 Thomas A, Necchi A, Muneer A, Tobias-Machado M, Tran ATH, Van Rompuy A-S, Spiess PE, Albersen M (2021) Penile Cancer. Nat Rev Dis Primers 7:1–24 Jacob JM et al (2019) mar., Comparative Genomic Profiling of Refractory and Metastatic Penile and Nonpenile Cutaneous Squamous Cell Carcinoma: Implications for Selection of Systemic Therapy., J. Urol. , vol. 201, n o 3, pp. 541–548 Huang J, Zhang L, Wan D, Zhou L, Zheng S, Lin S, Qiao Y (2021) Extracellular Matrix and Its Therapeutic Potential for Cancer Treatment. Signal Transduct Target Ther 6:153 Morishita K, Watanabe K, Ichijo H (2019) Cell Volume Regulation in Cancer Cell Migration Driven by Osmotic Water Flow. Cancer Sci 110:2337–2347 Ereño C, Gaafar A, Garmendia M, Etxezarraga C, Bilbao FJ, López JI (2008) Basaloid Squamous Cell Carcinoma of the Head and Neck: A Clinicopathological and Follow-up Study of 40 Cases and Review of the Literature. Head Neck Pathol 2:83–91 Gupta B, Bhattacharyya A, Singh A, Sah K, Gupta V (2018) Basaloid Squamous Cell Carcinoma – A Rare and Aggressive Variant of Squamous Cell Carcinoma: A Case Report and Review of Literature. Natl J Maxillofac Surg 9:64–68 Morishita K, Watanabe K, Ichijo eH (2019) Cell volume regulation in cancer cell migration driven by osmotic water flow. Cancer Sci 110:2337–2347n o 8 Stroka KM, Jiang H, Chen S-H, Tong Z, Wirtz D, Sun SX, Konstantopoulos K (2014) Water Permeat Drives Tumor Cell Migration Confined Microenvironments Cell 157:611–623 Oster GF, Perelson eAS (1987) The Physics of Cell Motility, J. Cell Sci. , vol. n o Supplement_8, pp. 35–54, 1987 Rosengren S, Henson PM, Worthen eGS (1994) Migration-associated volume changes in neutrophils facilitate the migratory process in vitro, Am. J. Physiol. Physiol. , vol. 267, n o 6, pp. C1623–C1632 Loitto V-M, Forslund T, Sundqvist T, Magnusson K-E, Gustafsson eM (2002) Neutrophil leukocyte motility requires directed water influx, J. Leukoc. Biol. , vol. 71, n o 2, pp. 212–222 Rosengren S, Henson PM, Worthen GS (1994) Migration-Associated Volume Changes in Neutrophils Facilitate the Migratory Process in Vitro. Am J Physiol 267:C1623–1632 Mehrabi M, Amini F, Mehrabi S (2018) Active Role of the Necrotic Zone in Desensitization of Hypoxic Macrophages and Regulation of CSC-Fate: A Hypothesis. Front. Oncol. , 8 Liu Z-G, Jiao D, Necroptosis (2019) Tumor Necrosis and Tumorigenesis. Cell Stress 4:1–8 Jain RK, Martin JD, Stylianopoulos T (2014) The Role of Mechanical Forces in Tumor Growth and Therapy. Annu Rev Biomed Eng 16:321–346 Penna LS, Henriques JAP, Bonatto eD (2017) Anti-mitotic agents: Are they emerging molecules for cancer treatment? Pharmacol. Ther. , vol. 173, pp. 67–82, maio Liu Y et al (jan. 2024) Developing a prognosis and chemotherapy evaluating model for colon adenocarcinoma based on mitotic catastrophe-related genes. Sci Rep 14:1655n o 1 Kriaucionis S, Tahiliani eM (2014) Expanding the epigenetic landscape: novel modifications of cytosine in genomic DNA., Cold Spring Harb. Perspect. Biol. , vol. 6, n o 10, p. a018630, out Ouderkirk JL, Krendel eM (2014) Non-muscle myosins in tumor progression, cancer cell invasion, and metastasis., Cytoskeleton (Hoboken). , vol. 71, n o 8, pp. 447–463, ago Gebrekiristos M, Melson J, Jiang A, Buckingham eL (jun. 2022) DNA methylation and miRNA expression in colon adenomas compared with matched normal colon mucosa and carcinomas. Int J Exp Pathol 103:74–82n o 3 Matthews Q, Jirasek A, Lum J, Duan X, Brolo eAG (2010) Variability in Raman spectra of single human tumor cells cultured in vitro: Correlation with cell cycle and culture confluency. Appl Spectrosc 64:871–887n o 8 Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4559053","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":316556688,"identity":"e6572fb8-3f91-4202-9e8e-9a36e2ace60e","order_by":0,"name":"Joel Félix Silva Diniz-Filho","email":"","orcid":"","institution":"Federal University of Maranhão","correspondingAuthor":false,"prefix":"","firstName":"Joel","middleName":"Félix Silva","lastName":"Diniz-Filho","suffix":""},{"id":316556692,"identity":"96683a3d-0f8b-4320-b7c5-2d6fa15cf1a1","order_by":1,"name":"Ana Caroline Muniz Silva","email":"","orcid":"","institution":"Federal University of Maranhão","correspondingAuthor":false,"prefix":"","firstName":"Ana","middleName":"Caroline Muniz","lastName":"Silva","suffix":""},{"id":316556694,"identity":"8edd62f9-28c8-492c-ae63-71d8f4d6e113","order_by":2,"name":"Antônio Augusto Lima Teixeira","email":"","orcid":"","institution":"Federal University of Maranhão","correspondingAuthor":false,"prefix":"","firstName":"Antônio","middleName":"Augusto Lima","lastName":"Teixeira","suffix":""},{"id":316556698,"identity":"02058b9c-6a66-4dd2-888e-c4a4a17dc087","order_by":3,"name":"Bruna Larissa Nolêto Sousa","email":"","orcid":"","institution":"Federal University of Maranhão","correspondingAuthor":false,"prefix":"","firstName":"Bruna","middleName":"Larissa Nolêto","lastName":"Sousa","suffix":""},{"id":316556702,"identity":"7ea43cf1-aa43-4089-9b5b-8ea8d337d364","order_by":4,"name":"Ralph Santos-Oliveira","email":"","orcid":"","institution":"Brazilian Nuclear Energy Commission","correspondingAuthor":false,"prefix":"","firstName":"Ralph","middleName":"","lastName":"Santos-Oliveira","suffix":""},{"id":316556703,"identity":"1f10bdd4-cadf-4241-9f4c-5334b0938c38","order_by":5,"name":"Gyl Eanes Barros Silva","email":"","orcid":"","institution":"Federal University of Maranhão","correspondingAuthor":false,"prefix":"","firstName":"Gyl","middleName":"Eanes Barros","lastName":"Silva","suffix":""},{"id":316556705,"identity":"c4a57cb0-7679-473b-bba8-81aef74059d7","order_by":6,"name":"Clenilton Costa dos Santos","email":"","orcid":"","institution":"Federal University of Maranhão","correspondingAuthor":false,"prefix":"","firstName":"Clenilton","middleName":"Costa dos","lastName":"Santos","suffix":""},{"id":316556707,"identity":"e72ca8bc-9c96-4cf7-a54d-5e10c2f8d37f","order_by":7,"name":"Luciana Magalhães Rebelo Alencar","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9ElEQVRIiWNgGAWjYBACPiA+AGaxQwTkGJiBfMYG3FrY4FqYIQLGDGwJhLUwIGtJbCCoRSL54YGfO+wY+JuZjz34UXMvfcMx7sQPjDvu4dGSZnCw90wyg8RhtnTDnmPFuRuO8W6WYDxTjEdLDsMB3jagqw7zmEkD/ZG74X7vBgnGtgS8Wg7+batnkD/M/02a4V9CugHQlh+EtBzmbTvMYHCYh00aqDIBqGUbflt4nhkclm07zmN4mM1MsrcvwXAmUItF4hncWvjZkx9/fNtWLSd3vPmZxI9vCfJ8QIfd+LgDtxYY4EHlEtYwCkbBKBgFowAfAABkN1AXRWwR2wAAAABJRU5ErkJggg==","orcid":"","institution":"Federal University of Maranhão","correspondingAuthor":true,"prefix":"","firstName":"Luciana","middleName":"Magalhães Rebelo","lastName":"Alencar","suffix":""}],"badges":[],"createdAt":"2024-06-10 15:44:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4559053/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4559053/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":59606526,"identity":"1054c746-12b1-43d6-9cad-f3543ef31336","added_by":"auto","created_at":"2024-07-03 18:53:05","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":686329,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOptical microscopy.\u003c/strong\u003e Representative optical microscopy images of tissues from the control group (non-tumorigenic) (A) and those with PCa: Basaloid (B) and Sarcomatoid (C). The black arrow indicates the outermost layer, the stratum corneum.\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4559053/v1/5e054b3a1aaad1c263f7777f.png"},{"id":59607047,"identity":"4d4a09af-3b6c-43c6-b3eb-37ae5f8ff1d5","added_by":"auto","created_at":"2024-07-03 19:01:05","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":358559,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAtomic Force Microscopy Maps.\u003c/strong\u003e 25 x 25 µm topographic AFM maps of the ultrastructure of tissues from the control group (non-tumorigenic) (A) and those with PCa: Basaloid (B) and Sarcomatoid (C) and its respective three-dimensional representations (D-F).\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4559053/v1/57f28e7a81eaa2d382e6dad2.png"},{"id":59606530,"identity":"93f50f3b-4712-4d4d-b287-eef6d299b4f0","added_by":"auto","created_at":"2024-07-03 18:53:06","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":629894,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePCa Surface porous.\u003c/strong\u003e Topographic AFM maps of the ultrastructure of tissues from Basaloid (A) and Sarcomatoid (B) tumors. Arrows point to the holes observed in each cancer subtype, and dotted circles delimit the holes in each tumor type.\u003c/p\u003e","description":"","filename":"Onlinefloatimage42.png","url":"https://assets-eu.researchsquare.com/files/rs-4559053/v1/dc451e9fe61e6d74cb9128d3.png"},{"id":59606529,"identity":"85f0e0d0-80ae-4dbd-bddf-0195a17410a6","added_by":"auto","created_at":"2024-07-03 18:53:05","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":65327,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAFM Quantitative data\u003c/strong\u003e. Quantitative data of ultrastructural properties of tissues from the control group (non-tumorigenic) and CAPe patients. (A) Roughness, (B) tissue Surface Area, (C) Volume, and (D) Seformation Scattering charts. The (*) indicates significant differences in the ANOVA test with Turkey for p\u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4559053/v1/78c1674e3f7ad9317accee37.png"},{"id":59606532,"identity":"d1f2be91-82ff-46ea-8ce0-49773029eb89","added_by":"auto","created_at":"2024-07-03 18:53:06","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":55682,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMolecular identification\u003c/strong\u003e. Average spectra and identification of vibrational modes related to the control group (black), sarcomatoid group (red), and basaloid group (blue). The red strips identify the bands corresponding to proteins and amino acids, the blue strips correspond to the lipid and carbohydrate modes, and the yellow strips correspond to other modes.\u003c/p\u003e","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-4559053/v1/875504a6bf53bb3a4cefd00c.png"},{"id":59606531,"identity":"93d15092-6268-425d-85c3-825233aa8653","added_by":"auto","created_at":"2024-07-03 18:53:06","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":50835,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePCA Analysis from Raman Spectra\u003c/strong\u003e. (A) Control x Sarcomatoid in LWN; (B) Control x Sarcomatoid in HWN; (C) Control x Basaloid in LWN; (D) Control x Basaloid in HWN (E) Sarcomatoid x Basaloid in LWN and (F) Sarcomatoid x Basaloid in HWN.\u003c/p\u003e","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-4559053/v1/cde3637f5fd009dd2acfb8a9.png"},{"id":61801693,"identity":"18dede32-0ada-4770-9aa2-f4e9fbb53021","added_by":"auto","created_at":"2024-08-05 17:48:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2511215,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4559053/v1/131174bd-c88e-4e6e-aad0-b6ac7f37f183.pdf"},{"id":59606528,"identity":"88c4de4c-f346-40fe-b13a-5cde266dfc54","added_by":"auto","created_at":"2024-07-03 18:53:05","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":248061,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGRAPHICAL ABSTRACT\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4559053/v1/36ced29a2df8c8a19483f0a5.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Penile Cancer: Innovations in Ultrastructural and Vibrational Markers","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003ePenile cancer (PCa) presents a significant geographic and socioeconomic disparity in its incidence rates. It is relatively rare in developed regions such as North America and Eastern Europe. In contrast, its prevalence is notably higher in developing countries across Asia, Africa, and South America [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Recent data indicate a concerning uptrend in PCa incidence in these areas, highlighting the disease's impact on public health and underscoring the need for targeted research and healthcare strategies to address this growing challenge [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHistologically, most penile squamous cell carcinomas (SCCs) share morphological similarities with squamous neoplasms originating in other organs, particularly closely resembling those found in oral and cervical regions. This commonality underscores a shared pathogenesis that might be rooted in squamous cell pathology [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. About 70% of penile SCCs are classified as the usual type, characterized by their histological conformity to the classic presentation of squamous cell carcinomas. The remaining 30% of cases are distributed among distinct histological subtypes, including basaloid, verrucous, papillary, and sarcomatoid variants [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Each of these histological subtypes exhibits unique clinical behaviors and prognostic implications.\u003c/p\u003e \u003cp\u003eWithin the spectrum of penile squamous cell carcinoma (SCC) subtypes, the basaloid and sarcomatoid variants merit specific focus due to their unique histopathological characteristics and clinical implications. Basaloid SCC, which represents about 10% of penile SCC occurrences, is more aggressive. This subtype is defined by the formation of small cellular clusters exhibiting minimal cytoplasm, pronounced nuclei, and a high frequency of mitotic figures, frequently in necrotic tissue. The designation \"basaloid\" reflects the morphological similarity of these neoplastic cells to basal cells. Histologically, basaloid SCC is identified by a distinctive architectural feature: a palisade-like configuration of cells at the periphery of tumor islands, coupled with a notable lack of intercellular bridges and the presence of central coagulative necrosis, often referred to as comedonecrosis. These defining characteristics contribute to the diagnostic criteria for basaloid SCC and underscore the subtype's aggressive nature and potential impact on patient prognosis [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Basaloid squamous cell carcinoma (SCC) is characterized by its intimate interaction with the surrounding connective stroma, which supports clusters of basaloid cells. This interaction manifests as distinctive slits or interfaces, commonly referred to as the epithelium-stroma interface. These morphological features facilitate the carcinoma's identification and aggressive behavior and frequently demonstrate a propensity for deep infiltration into the underlying tissues. A significant clinical concern with basaloid SCC is its high rate of lymphatic spread; more than half of the patients exhibit involvement of the inguinal lymph nodes at the time of initial diagnosis. This pattern of aggressive growth and early lymphatic involvement underscores the importance of prompt, accurate diagnosis and comprehensive management strategies to address both the primary tumor and potential metastatic disease [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. This subtype of PaC is directly associated with HPV.\u003c/p\u003e \u003cp\u003eHuman papillomavirus (HPV) represents a pivotal factor in the etiology of penile cancer, a malignancy that, although relatively uncommon globally, poses significant health challenges in various regions, especially in developing countries. HPV, a DNA virus from the Papillomaviridae family, is known for infecting epithelial cells of the skin and mucous membranes, leading to a range of outcomes from benign lesions to malignancies [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The oncogenic potential of HPV, particularly high-risk subtypes such as HPV 16 and 18, is linked to their genetic material integrating into the host cell's DNA, thereby disrupting normal cell cycle regulation and promoting malignant transformation [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. This process is mediated by the viral oncoproteins E6 and E7, which interfere with tumor suppressor proteins p53 and retinoblastoma (Rb) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAlthough rare (occurring in less than 1% of cases), the sarcomatoid subtype demonstrates significant biological aggressiveness, attaining considerable sizes and penetrating deeply into adjacent structures. Microscopically, it is characterized by spindle-shaped cells intermingled with cells of bizarre or giant shapes and may present sarcomatous components, such as chondrosarcoma or osteosarcoma. The histology of sarcomatoid SCC is biphasic, involving the differentiation of squamous epithelial and mesenchymal components. In the transformation process from squamous cells to spindle cells, the epitheliomesenchymal transition occurs through a decrease in the expression of E-cadherin, the primary epithelial intercellular adhesion molecule, and an increase in the expression of N-cadherin, responsible for the mobile phenotype of the cells [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe limited evidence on penile squamous cell carcinoma with epithelioid features (PCa) underscores a critical gap in our comprehensive understanding of its tumor biology. To bridge this knowledge gap, this study integrates Atomic Force Microscopy (AFM) and Raman Vibrational Spectroscopy (RS) as investigative tools for a detailed ultrastructural and molecular examination of PCa tumors, specifically focusing on sarcomatoid and basaloid subtypes. The juxtaposition of ultrastructural and molecular data from these advanced imaging techniques promises to shed light on the complex biology of penile squamous cell carcinoma, potentially paving the way for new approaches to its unique pathological features.\u003c/p\u003e"},{"header":"METHODOLOGY","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eTissue Selection\u003c/h2\u003e \u003cp\u003eSpecialized researchers collected the samples from three reference hospitals located in S\u0026atilde;o Lu\u0026iacute;s, Maranh\u0026atilde;o (Presidente Dutra University Hospital, Aldenora Bello Cancer Hospital, and Maranh\u0026atilde;o Cancer Hospital Dr. Tarqu\u0026iacute;nio Lopes Filho). Participants were informed about the study's research objectives, risks, and expected impacts. All participants signed the Informed Consent Form (ICF), which the Human Research Ethics Committee approved. Subjects who agreed to participate in the research were interviewed for socio-behavioral data collection using a data collection instrument. In contrast, those who did not agree were assured that there would be no harm to conventional hospital treatment and follow-up. After the material was collected for research, all materials were identified using a specific project code to ensure participants' confidentiality and privacy rights.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eInclusion Criteria\u003c/h2\u003e \u003cp\u003eThis study considered men over 18 years of age with clinical and anatomopathological diagnoses of penile cancer who had an amputation as the first therapeutic option. Only those who agreed to participate in the study by signing the Informed Consent Form (ICF) were included.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eExclusion Criteria\u003c/h2\u003e \u003cp\u003eThose who had undergone chemotherapy or radiotherapy before the surgical procedure were excluded.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eReview of Histological Slides and Selection of Study Area\u003c/h2\u003e \u003cp\u003eAfter applying inclusion and exclusion criteria, cases were selected, and their Hematoxylin and Eosin (H\u0026amp;E) stained slides underwent a reevaluation to confirm histological diagnosis and tumor classification according to the criteria proposed by the American Joint Committee on Cancer (AJCC) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The slide review was conducted independently by two pathologists. Tumor subclassification (CECs) was based on criteria established in the medical literature [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eTissue Collection\u003c/h2\u003e \u003cp\u003eThe samples used in this study were collected in the surgical center: the physician responsible for the amputation collected small fragments of fresh tissue containing tumor and non-tumor (normal) samples. The samples were stored in the following solutions: (1) RNAlater (ThermoFisher TM) for DNA extraction and HPV detection and (2) 10% buffered formalin for biophysical analyses.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eHPV Detection and Genotyping\u003c/h2\u003e \u003cp\u003eThe QIAamp Fast DNA Tissue kit (Qiagen, Cat. No. 51404) was used for DNA extraction. The extracted samples were evaluated for extraction quality by quantifying the total material on a NanoDrop spectrophotometer (ThermoFisher TM), with concentrations expressed in ng/\u0026micro;L, and purity assessment with 260/280 nm measurements (between 1.8 and 2.0) and 260/230 (above one). The samples were stored at -20\u0026deg;C until used in subsequent steps.\u003c/p\u003e \u003cp\u003eHPV detection was conducted by conventional PCR (Polymerase Chain Reaction) in two stages (nested PCR). In the first PCR, a set of generic primers called PGMY09/11, described by Gravitt et al. [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], which produced a 450 bp fragment of the HPV capsid L1 region, was used. The primer GP5+/6\u0026thinsp;+\u0026thinsp;was used in the second PCR, generating a 170 bp amplicon corresponding to the viral capsid L1 region. A pair of primers for the β-globin gene (366 bp fragment) was used as a positive control for the reaction. The final mix was 25 \u0026micro;L for each sample, using the MASTERMIX PCR PLATINUM SUPERFI kit (Life TechnologiesTM), followed by 45 cycles of 94\u0026deg;C for 45 seconds, 40\u0026deg;C for 1 minute, and 72\u0026deg;C for 1 minute; and finally 72\u0026deg;C for 10 minutes. The amplicons were separated on a 1.5% agarose gel and subjected to a constant voltage of 90V for 40 minutes. Only those with amplification for the β-globin and GP5+/6\u0026thinsp;+\u0026thinsp;genes were considered positive. Capillary electrophoresis sequenced positive cases, and their sequences were compared to those available in genetic databases using BLAST (Basic Local Alignment Search Tool) for viral genotyping.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eTissue Preparation\u003c/h2\u003e \u003cp\u003eThe PCa samples will be embedded in paraffin and cut using an ultramicrotome (model LEICA EM UC6), producing 2 \u0026micro;m thick sections. The biopsies will be deposited on 13 mm diameter glass slides and subsequently placed in an oven at 60 degrees for 30 minutes for dewaxing. After this process, the samples will be submerged in 30 ml of Xylene and gently shaken for 15 minutes, with 30 ml of Xylene being changed every 5 minutes. Subsequently, the samples will be rehydrated through a sequence of 90%, 80%, and 70% PA ethyl alcohol for tissue rehydration.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003eAtomic Force Microscopy Setup\u003c/h2\u003e \u003cp\u003eThe analysis by Atomic Force Microscopy was conducted using an AFM Multimode 8 (Bruker, Santa Barbara, CA, USA) in PeakForce Quantitative Nanomechanics mode - QNM. For this purpose, probes of the qp-HBC model (NanoSensors) with a nominal cantilever spring constant of 0.5 N/m and a tip radius smaller than 10 nm were utilized. All data were obtained with a scanning rate of 0.5 Hz and a curve acquisition frequency of 0.5 kHz. Three non-tumorous samples (Control Group), three samples of the sarcomatoid subtype, and three samples of the basaloid subtype were used. Each sample underwent 15 scans at distinct points. In total, 45 maps of 25\u0026micro;m x 25\u0026micro;m were analyzed for each group. Each scan contained 65536 force curves, providing a broad database for comparative analysis among the studied groups.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eUltrastructural Analysis\u003c/h2\u003e \u003cp\u003eFor AFM data, the statistical roughness analysis was based on the height of each pixel in the image, analyzed from the height map, according to the methodology described by Rates et al. [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Area and volume data were calculated from topographic maps of 25 x 25 \u0026micro;m of tissue surface (control/tumor) using the Gwyddion 2.57 software. The deformation parameter was calculated using the ratio \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(A/V\\)\u003c/span\u003e\u003c/span\u003e. According to Degiorgio et al. [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], changes in surface area and volume are related to changes in cell (tissue) membrane composition and membrane (tissue) component arrangements about changes in the cell\u0026acute;s surface area and volume and, consequently, in cell (tissue) deformability. Hole diameters for basaloid (n\u0026thinsp;=\u0026thinsp;669) and sarcomatoid tissue (n\u0026thinsp;=\u0026thinsp;94) were calculated using Gwyddion 2.57 software, applying the threshold edge detection data tool. Values were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eRaman Spectroscopy Analysis\u003c/h2\u003e \u003cp\u003eRaman Spectroscopy was used to analyze and identify the spectral differences obtained from the control group and groups of patients with PCa, previously disclosed through the clinical method. The measurements were carried out on Horiba\u0026rsquo;s T64000 spectrometer with a CCD (Charge Coupled Device) detection system cooled with liquid nitrogen. All measurements were obtained in backscatter geometry. The 532 nm line was used as a traction source with its maximum power for the measurements. The sample surface was viewed using a specific Olympus brand with an attached video camera. To focus the brightness on the surface, we used a 100\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\times\\)\u003c/span\u003e\u003c/span\u003e lens. Nine acquisitions were carried out with times of 20 seconds. The spectral region observed in our experiments was divided into intervals of 750 to 1750 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(c{m}^{-1}\\)\u003c/span\u003e\u003c/span\u003e (Low Wavenumbers - LWN) and 2650 to 3150 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(c{m}^{-1}\\)\u003c/span\u003e\u003c/span\u003e (High Wavenumbers - HWN)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eSpectral Pre-Processing\u003c/h2\u003e \u003cp\u003eData processing was conducted using LabSpec6 software. The narrow peaks caused by cosmic rays were sequentially removed, and the variable fluorescence background and the glass substrate were estimated using a fifth-order polynomial fitting and subsequently subtracted. Each spectrum was smoothed using a polynomial smoothing algorithm before analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003ePrincipal Component Analysis - PCA\u003c/h2\u003e \u003cp\u003ePrincipal Component Analysis (PCA) was applied to the spectral dataset, a statistical analysis method capable of reducing the dimensionality of the data while capturing most of the variation in the original dataset. The spectra were analyzed following the methodology of Yi Hong Ong and coworkers [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], where variance analysis was employed on the scores of the first ten principal components to determine which PC exhibited significant differences in mean scores between the two groups of cells, utilizing OriginLab software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eStatistical test following a single criterion was evaluated using ANOVA and Tukey\u0026rsquo;s post-test, considering the values were statistically significant when p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Statistical analyses and graphics were performed using the ORIGIN software. The calculated error was the standard deviation (SD) in all data.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003eFigure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows a representative optical microscopy image of each tissue analyzed. In Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, representing the control group, the histological slide shows the layers of the epidermis with stratified squamous cells, without neoplastic changes, with the stratum corneum being the outermost layer with a pink color, as shown by the arrow, and the basal layer being the innermost with large, elongated and hyperchromatic nuclei pigmented with hematoxylin [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB shows the basaloid group, indicated by the abnormal growth of bluish, small, uniform cells with round nuclei and scant cytoplasm, which resemble the basal cells of epithelial tissue, normally presenting a palisade arrangement in the peripheral cells of the tumor islets, absence of intercellular bridges and the presence of central coagulation necrosis [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC represents the sarcomatoid group, marked by the differentiation of squamous and mesenchymal components, characterized by the expression of spindle-shaped sarcomatous cells, which exhibit atypical and elongated nuclei, resulting from the epitheliomesenchymal transition, in which the squamous cell is transformed into spindle cells [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Such histopathological components show the conformational changes undergone by the pathological tissue compared to the control group.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe representative high-resolution AFM maps of each group reveal ultrastructural changes on the surface of PCa tissues, as observed in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA shows a 25 \u0026micro;m\u003csup\u003e2\u003c/sup\u003e scan over a non-tumor region of penile tissue and its respective 3D view in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, compatible with the preserved ultrastructural morphology of the stratum spinosum layer [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In contrast, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE 3D view) shows a scan of the same size for basaloid cancer tumor tissue associated with HPV infection [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The submicrometric-sized holes in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB may be related to the percolation or diffusion of viral particles in this type of tumor. In PCa sarcomatoid subtype tissue, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF 3D view), several stretches of tissue form micrometric holes on its surface. This fact is associated with high vascularity in cancer tissues, indicating rapid tumor growth, as vascularization is necessary to supply nutrients and oxygen to cancer cells [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, one can see how each PCa subtype promotes fenestrations in the tumor tissue. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA shows a representative image of the basaloid subtype, showing uniform fenestrations (or pores) of submicron diameter (0.688\u0026thinsp;\u0026plusmn;\u0026thinsp;0.053 \u0026micro;m) (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD). As with many medications [\u003cspan additionalcitationids=\"CR29 CR30\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], which prevent or reduce its effectiveness, viral particles can become trapped by this complex porous structure, which may associate this subtype with HPV infection. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB shows a representative image of the surface detail of the sarcomatoid subtype tumor tissue. Here, it is possible to observe a greater presence of dark regions (holes), micrometric in size (6.39\u0026thinsp;\u0026plusmn;\u0026thinsp;1.28 \u0026micro;m), compatible with tissue failures. These holes have a medium diameter compatible with capillaries that irrigate the tumor tissue [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. When the vascular and nutritional supply aligned with the high mitotic activity of the tumors does not supply the demands of the tumor microenvironment, the formation of foci of necrosis, typical in basaloid and sarcomatoid PCa, occurs, visible through the stretches [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Furthermore, high vascularity can also facilitate the spread of cancer to other parts of the body through the bloodstream. This stretching may also be linked to the cytoskeleton and extracellular matrix collagen\u0026rsquo;s structural changes that gradually promote cancer progression [\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMotivated by these qualitative ultrastructural differences observed in tumor tissues concerning non-tumor tissue and also between the different types of tumors (basaloid and sarcomatoid), we analyzed quantitative ultrastructural parameters of the groups, such as mean quadratic roughness of the tissue surface, surface area, tissue image volume and deformation (A/V ratio). The results can be seen in the panel shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. The scatterplot shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA presents the mean squared roughness results for each group analyzed. The mean values ​​and their respective standard deviations are 283.8\u0026thinsp;\u0026plusmn;\u0026thinsp;8.2 nm, 360.9\u0026thinsp;\u0026plusmn;\u0026thinsp;12.3 nm, and 284.1\u0026thinsp;\u0026plusmn;\u0026thinsp;8.1 nm for the control, basaloid, and sarcomatoid tissues. A greater number of holes (fenestrations) in the basaloid tumor tissue is reflected in the increased roughness result, which may be associated with the greater capacity of these tumors to trap a greater quantity of viral particles [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB shows the scatter plot of surface area values ​​of tissues from each group analyzed. The average values ​​obtained were 810.4\u0026thinsp;\u0026plusmn;\u0026thinsp;10.1 \u0026micro;m\u003csup\u003e2\u003c/sup\u003e, 738.2\u0026thinsp;\u0026plusmn;\u0026thinsp;4.6 \u0026micro;m\u003csup\u003e2\u003c/sup\u003e, and 767.4\u0026thinsp;\u0026plusmn;\u0026thinsp;5.7 \u0026micro;m\u003csup\u003e2\u003c/sup\u003e, respectively for the control, basaloid and sarcomatoid groups. Here, it is possible to observe a trend, with statistical relevance, of a decrease in the surface area of ​​tumor tissues when compared with non-tumor ones.\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC presents the volume results of the maps obtained from the analyzed tissues. The average values ​​obtained were 940.8\u0026thinsp;\u0026plusmn;\u0026thinsp;38.0, 656.4\u0026thinsp;\u0026plusmn;\u0026thinsp;29.4, and 395.2\u0026thinsp;\u0026plusmn;\u0026thinsp;18.5 \u0026micro;m\u003csup\u003e3\u003c/sup\u003e for the control, basaloid and sarcomatoid groups, respectively. As with the surface area, the maps obtained from the tumor tissue samples showed a reduced average volume compared to the control group.\u003c/p\u003e \u003cp\u003eThe graph shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD shows the scatter plot of surface deformation calculated from the geometric parameters of the images obtained from each group. The average values ​​found were, respectively, 0.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03, 1.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04, and 2.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 \u0026micro;m\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for the control, basaloid and sarcomatoid groups. It is possible to observe greater deformability in tumor samples than in non-tumor ones. This fact may be associated with the greater capacity for tumor deformation at the cellular level [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] since we are analyzing the ultrastructure of tumor tissues, which enables greater invasion of these tumors into new sites.\u003c/p\u003e \u003cp\u003eAnother promising approach is investigating the vibrational signature obtained through Raman Spectroscopy in the tissues examined. This biochemical study, combined with AFM data, may bring new perspectives to the study of penile tumors. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e shows the average spectrum obtained from 30 samples from each group. The bands refer to the vibrational modes associated with the main biochemical components of tissues, such as proteins and amino acids, carbohydrates, and lipids, among others, for both low and high wavelengths. This way, it is possible to observe the differences in the biochemical composition among the tissues analyzed.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe specific wavelengths for each identified mode can be found in Table\u0026nbsp;1.\u003c/p\u003e \u003cp\u003e\u003cstrong\u003eTable 1:\u003c/strong\u003e Assignments of each mode of the tissue Raman spectrum [41\u0026ndash; 46].\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"501\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.2%\"\u003e\n \u003cp\u003eWavenumber (cm\u003csup\u003e-1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.8%\" valign=\"top\"\u003e\n \u003cp\u003eAmino acid/ Protein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.2%\" valign=\"top\"\u003e\n \u003cp\u003eLipid/ Carbohydrate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.8%\" valign=\"top\"\u003e\n \u003cp\u003eOther\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.2%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.8%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.2%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.8%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.2%\" valign=\"top\"\u003e\n \u003cp\u003e813\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.8%\" valign=\"top\"\u003e\n \u003cp\u003eC \u0026ndash; C str.\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.2%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.8%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.2%\" valign=\"top\"\u003e\n \u003cp\u003e856\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.8%\" valign=\"top\"\u003e\n \u003cp\u003eProline\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.2%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.8%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.2%\" valign=\"top\"\u003e\n \u003cp\u003e874\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.8%\" valign=\"top\"\u003e\n \u003cp\u003eC \u0026ndash; C str.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.2%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.8%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.2%\" valign=\"top\"\u003e\n \u003cp\u003e888\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.8%\" valign=\"top\"\u003e\n \u003cp\u003eProtein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.2%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.8%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.2%\" valign=\"top\"\u003e\n \u003cp\u003e919\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.8%\" valign=\"top\"\u003e\n \u003cp\u003eProline\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.2%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.8%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.2%\" valign=\"top\"\u003e\n \u003cp\u003e937\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.8%\" valign=\"top\"\u003e\n \u003cp\u003eProline\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.2%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.8%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.2%\" valign=\"top\"\u003e\n \u003cp\u003e1002\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.8%\" valign=\"top\"\u003e\n \u003cp\u003ePhenylalanine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.2%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.8%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.2%\" valign=\"top\"\u003e\n \u003cp\u003e1031\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.8%\" valign=\"top\"\u003e\n \u003cp\u003ePhenylalanine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.2%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.8%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.2%\" valign=\"top\"\u003e\n \u003cp\u003e1058\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.8%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.2%\" valign=\"top\"\u003e\n \u003cp\u003eLipids\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.8%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.2%\" valign=\"top\"\u003e\n \u003cp\u003e1100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.8%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.2%\" valign=\"top\"\u003e\n \u003cp\u003eLipids\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.8%\" valign=\"top\"\u003e\n \u003cp\u003eFatty acid\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.2%\" valign=\"top\"\u003e\n \u003cp\u003e1131\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.8%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.2%\" valign=\"top\"\u003e\n \u003cp\u003ePhospholipids\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.8%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.2%\" valign=\"top\"\u003e\n \u003cp\u003e1159\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.8%\" valign=\"top\"\u003e\n \u003cp\u003eC \u0026ndash; C / C \u0026ndash; N str.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.2%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.8%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.2%\" valign=\"top\"\u003e\n \u003cp\u003e1169\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.8%\" valign=\"top\"\u003e\n \u003cp\u003eProline\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.2%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.8%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.2%\" valign=\"top\"\u003e\n \u003cp\u003e1207\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.8%\" valign=\"top\"\u003e\n \u003cp\u003eHydroxyproline, Tyrosine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.2%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.8%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.2%\" valign=\"top\"\u003e\n \u003cp\u003e1239 \u0026ndash; 1272\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.8%\" valign=\"top\"\u003e\n \u003cp\u003eAmide III\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.2%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.8%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.2%\" valign=\"top\"\u003e\n \u003cp\u003e1293\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.8%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.2%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.8%\" valign=\"top\"\u003e\n \u003cp\u003eCytosine\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.2%\" valign=\"top\"\u003e\n \u003cp\u003e1315\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.8%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.2%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.8%\" valign=\"top\"\u003e\n \u003cp\u003eGuanine\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.2%\" valign=\"top\"\u003e\n \u003cp\u003e1340\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.8%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.2%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.8%\" valign=\"top\"\u003e\n \u003cp\u003eNucleic Acid\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.2%\" valign=\"top\"\u003e\n \u003cp\u003e1381\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.8%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.2%\" valign=\"top\"\u003e\n \u003cp\u003eCH\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.8%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.2%\" valign=\"top\"\u003e\n \u003cp\u003e1392\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.8%\" valign=\"top\"\u003e\n \u003cp\u003eC \u0026ndash; N str.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.2%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.8%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.2%\" valign=\"top\"\u003e\n \u003cp\u003e1402\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.8%\" valign=\"top\"\u003e\n \u003cp\u003eMethyl ben groups.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.2%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.8%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.2%\" valign=\"top\"\u003e\n \u003cp\u003e1416\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.8%\" valign=\"top\"\u003e\n \u003cp\u003eC = C str.\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.2%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.8%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.2%\" valign=\"top\"\u003e\n \u003cp\u003e1450\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.8%\" valign=\"top\"\u003e\n \u003cp\u003eCH\u003csub\u003e2\u003c/sub\u003e ben.\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.2%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.8%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.2%\" valign=\"top\"\u003e\n \u003cp\u003e1458\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.8%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.2%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.8%\" valign=\"top\"\u003e\n \u003cp\u003eNucleic Acid\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.2%\" valign=\"top\"\u003e\n \u003cp\u003e1514\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.8%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.2%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.8%\" valign=\"top\"\u003e\n \u003cp\u003eCytosine\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.2%\" valign=\"top\"\u003e\n \u003cp\u003e1638 \u0026ndash; 1665\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.8%\" valign=\"top\"\u003e\n \u003cp\u003eAmide I\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.2%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.8%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.2%\" valign=\"top\"\u003e\n \u003cp\u003e2728\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.8%\" valign=\"top\"\u003e\n \u003cp\u003eC \u0026ndash; H str.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.2%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.8%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.2%\" valign=\"top\"\u003e\n \u003cp\u003e2853\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.8%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.2%\" valign=\"top\"\u003e\n \u003cp\u003eCH\u003csub\u003e2\u003c/sub\u003e sym. str.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.8%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.2%\" valign=\"top\"\u003e\n \u003cp\u003e2888\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.8%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.2%\" valign=\"top\"\u003e\n \u003cp\u003eCH\u003csub\u003e2\u003c/sub\u003e asym. str.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.8%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.2%\" valign=\"top\"\u003e\n \u003cp\u003e2935\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.8%\" valign=\"top\"\u003e\n \u003cp\u003eCH\u003csub\u003e3\u003c/sub\u003e sym. str.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.2%\" valign=\"top\"\u003e\n \u003cp\u003eCH\u003csub\u003e3\u003c/sub\u003e sym. str.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.8%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.2%\" valign=\"top\"\u003e\n \u003cp\u003e2960 \u0026ndash; 2980\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.8%\" valign=\"top\"\u003e\n \u003cp\u003eCH\u003csub\u003e3\u003c/sub\u003e asym. str.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.2%\" valign=\"top\"\u003e\n \u003cp\u003eCH\u003csub\u003e3\u003c/sub\u003e asym. str.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.8%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.2%\" valign=\"top\"\u003e\n \u003cp\u003e3008\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.8%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.2%\" valign=\"top\"\u003e\n \u003cp\u003e= CH str.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.8%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.2%\" valign=\"top\"\u003e\n \u003cp\u003e3030\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.8%\" valign=\"top\"\u003e\n \u003cp\u003eAromatic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.2%\" valign=\"top\"\u003e\n \u003cp\u003eAromatic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.8%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"22.2%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.8%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.2%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.8%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eAbbreviation: str. = stretching, sym. = symmetric, asym. = asymmetric, def. = deformation, ben. = bending.\u003c/p\u003e\n\u003cp\u003eTo evaluate the discriminatory capacity of the method used through multivariate analysis, Principal Component Analysis (PCA) was performed on all data contained in the Low Wavenumber (LWN) and High Wavenumber (HWN) regions). In total, 30 spectra from each sample group were analyzed, suitable for statistical analysis. The ellipses present in the graph delimit the area in which 95% of the data is included. In Figure 6, the first three main components are highlighted, which result in good total variability of the data set.\u003c/p\u003e\n\u003cp\u003eFigure 6A shows the confidence ellipses for the control and sarcomatoid groups considering the spectrum region between 750-1750 cm\u003csup\u003e-1\u003c/sup\u003e (low wavelengths - LWL). The first three main components add up to 81.9% confidence. Figure 6B shows the relationship between the same groups, now for high wavelengths (HWL), between 2650-3150 cm\u003csup\u003e-1\u003c/sup\u003e. In this spectrum range, the first three PCs total 97.2% confidence. Figure 5C shows the confidence ellipses for the control and basaloid groups for LWL, with a confidence of 82.3% for the first three PCs. In contrast, Figure 6D shows the relationship between the same groups for HWL, with a confidence of 95.4 % for the first three PCs. Figures 6E and F show the relationship between the tumor groups (basaloid and sarcomatoid) for LWL (76.2%) and HWL (96.3%), respectively.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe results suggest that cytoskeleton-induced modifications can change tumor tissue morphology. Sarcomatoid PCa has a biphasic character, marked by a squamous component with sarcomatous differentiation of spindle cells [14]. This epitheliomesenchymal transition is characterized by decreased expression of E-cadherin and increased expression of N-cadherin, which is responsible for the mobile phenotype of the cells [14]. Cadherins are polypeptides responsible for epithelial intercellular adhesion, associated with a group of catenin proteins that bind the actinic microfilaments of the cytoskeleton [47].\u003c/p\u003e\n\u003cp\u003eZemła and coworkers demonstrated that the most rigid conformations within the cell surface are made up of actin filaments, and the structural disarrangements in the organization of the cytoskeleton were attributed to lower cellular rigidity, giving a mobile aspect to cancer, which correlates with the modulations caused by losses of E-actin-bound cadherins [48]. Changes in cytoskeletal dynamics, mediated by changes in cadherin expression, can influence cell morphology, indicated by changes in area and volume, which express a reduction in volume and area data in the sarcomatoid group.\u003c/p\u003e\n\u003cp\u003eAnother possible explanation to describe the structural and functional changes of the actin cytoskeleton is L-plastin, a group of actin-bridging proteins that contribute to tumor cell invasion in a phosphorylation-dependent manner [35]. Phosphorylation of L-plastin at its Ser5 residue increases its ability to interact with actin, thus influencing its intracellular localization [35]. The supply of energy to trigger phosphorylation on the L-plastin residue may be associated with glycolytic enzymes, which in cancer cells, due to the high rate of glycolysis, are increased, producing ATP in the vicinity of the cytoskeleton through reversible binding of glycolytic enzymes to the cytoskeleton [51].\u003c/p\u003e\n\u003cp\u003ePCa has subtypes associated with HPV, such as basaloid, and non-associated subtypes, such as sarcomatoid [2]. HPV-related penile carcinogenesis, typical of the basaloid subtype, arises from the overexpression of the viral oncoproteins E6 and E7, causing cell cycle dysregulation and genomic instability [38]. The viral oncoprotein E6 interferes with the p53 pathway, a tumor suppressor protein, inhibiting apoptosis by targeting the protein for degradation. The inhibition of p53 by E6 promotes exacerbated cell proliferation and tumor cell immortalization. However, non-HPV-associated carcinogenesis, such as in the sarcomatoid subtype, may result from mutagenic changes in tumor suppressor genes [49]. In a study carried out by Jacob et al., patients with metastatic penile cancer had mutations in TP53 [50]. They were negative for HPV, suggesting that mutations in TP53 and consequent overexpression of p53 were associated with metastasis in patients with advanced cancer and decreased patient survival [2]. The P53 pathway is a regulator in the formation of tumor-associated collagen signature 3, a collagen bundle angled at 60\u0026deg; to 90\u0026deg; to the edge of the cancer and is indicated by cancer proliferation and invasion [36]. The changes show that the sarcomatoid subtype presents a specificity in the expression of collagen in the extracellular matrix that can alter its nanomechanical and ultrastructural properties, such as the reduction in area and volume, associated with ECM modulations that favor the formation of apertures.\u003c/p\u003e\n\u003cp\u003eThe modification in surface roughness may be associated with biological processes underlying cancer development, such as uncontrolled cell proliferation and reorganization of the ECM [51]. Metastatic cancer cells exhibit an expanded expression of transport proteins such as ion channels, ion transporters, and aquaporins. These ion/water transport proteins, such as NHE1, NKCC1, AE2, ENaC, AQPs, IK channel, VRACs, ClC-3, and TMEM16s, often demonstrate elevated activity or expression in cancer cells. The increase in expression of these membrane proteins may justify the roughness observed in basaloid and sarcomatoid PCa, indicating a possible adaptation of cancerous tissues for more effective and invasive dissemination in other tissues [52].\u003c/p\u003e\n\u003cp\u003eThere was a reduction in the surface area of PCa tissues, which was more evident in the basaloid subtype, affected by the HPV virus. When comparing the samples with the control group, a similar pattern can be seen in the maps, with a decrease in height between the sarcomatoid and basaloid samples. This reduction in surface area suggests greater aggressiveness of the tumor, especially in the basaloid and sarcomatoid subtypes, classified as aggressive and with a high rate of nodal metastasis [53,54].\u003c/p\u003e\n\u003cp\u003eCompared to control samples, the analysis of basaloid and sarcomatoid samples revealed a reduction in volume. Transport proteins, such as ion channels, ion transporters, and aquaporins (AQPs), regulate cell volume during exposure to osmotic stress [55]. Studies indicate that water flow related to osmotic gradients generated by ionic transport contributes to cell migration [56 \u0026ndash; 59]. It was reported that cell migration is attenuated by extracellular hypertonicity; cell shrinkage, which inhibits local volume, would facilitate cell migration [60]. Furthermore, the osmotic gradient is responsible for regulating the expression of ion/water transport proteins and their changes in location in the membrane, modulating the cycles of protrusion of the leading edge and retraction of the rear part of the cell during migration [61]. These aspects demonstrate that changes in protein expression in metastatic cells, altered by extracellular osmotic stress, directly impact cell migration, typical of metastatic cancer. The correlation with volume and deformation data shows that changes in cellular structure caused by the osmolarity of the medium can impact the reduction of metastatic cell volume and increase cellular deformation of the basaloid and sarcomatoid subtypes compared to the control group.\u003c/p\u003e\n\u003cp\u003eThe rapid growth of cancer cells can exceed blood supply capacity, leading to areas of necrosis due to a lack of oxygen and nutrients [62,63]. Compression of surrounding blood vessels can result in atrophy [64]. Both cancer subtypes, basaloid and sarcomatoid, show high rates of mitosis and areas of necrosis, with basaloid characterized by comedonecrosis. Excessive mitotic activity concerning vascular and nutritional supply can result in tissue necrosis [65, 66], influencing the area and volume measurements observed in AFM. Hypoxic conditions in the collagen-rich ECM, intensified by the interaction between cancer cells and collagen, affect vascular supply. Factors such as HIF-1, LOX, and metalloproteinase play roles in this process, as they are related to cancerous blood vessels. The firmness of collagen in the matrix affects vascular growth, impacting the formation of necrotic foci and fissures identified in AFM [50]. The increase in deformation in the basaloid and sarcomatoid groups can be explained by the same mechanisms, given that the deformation is directly correlated with area and volume.\u003c/p\u003e\n\u003cp\u003eRaman spectroscopy analysis strongly corroborates these data. When a molecular group changes, the vibrational modes relative to it are also changed. Changes in the intensity, position, and broadening of the Raman spectrum peak can verify this fact. The greatest discrepancies between control penis tissues and tissues appear in modes located in the low wavenumber range (LWN) 700 to 1800 cm\u003csup\u003e-1\u003c/sup\u003e, where vibrational modes related to proteins are observed, such as the of Proline (919 cm\u003csup\u003e-1\u003c/sup\u003e), amino acids are essential amino acids that are abundant in structural proteins like collagen, and stretching of the C \u0026ndash; H and C = C bonds (1392 cm\u003csup\u003e-1\u003c/sup\u003e and 1416 cm\u003csup\u003e-1\u003c/sup\u003e respectively). In the range between 1239 and 1272 cm\u003csup\u003e-1\u003c/sup\u003e), attributed to Amide III and in the region 1638 to 1665 cm\u003csup\u003e-1\u003c/sup\u003e, attributed to Amide I, which are groups composed of carbon, oxygen, and nitrogen atoms (CONH), plays a crucial role in the formation of proteins. These bonds are essential for conferring structural rigidity and provide information about secondary structure organization in PCa tissues.\u003c/p\u003e\n\u003cp\u003eFurthermore, changes were identified in the bands corresponding to lipids (1131 and 1381 cm\u003csup\u003e-1\u003c/sup\u003e). These modes reflect the composition, organization, and structure of lipids in penile cancer tissues, providing valuable information about the tissue\u0026rsquo;s biochemistry. The presence of cytosine (1514 cm\u003csup\u003e-1\u003c/sup\u003e) in PCa tissues indicates how mutations or epigenetic changes can be critical in transforming a normal cell into a cancerous cell [67 \u0026ndash; 68].\u003c/p\u003e\n\u003cp\u003eIn the high wavenumber region (HWN) of the Raman spectrum, between 2700 and 3100 cm\u003csup\u003e-1\u003c/sup\u003e), stretch bands of the C \u0026ndash; H bonds of lipids present in the membranes of PCa tissues are detected, described by Matthews et al. [69]. Despite not demonstrating the precursor of lipid breakdown, this result shows that these components are most likely related to carcinogenic transformation. These vibrations also provide crucial information about the composition and organization of lipids in the lipid layers of PCa tissues, playing a fundamental role in membrane integrity and permeability. In the same way, as in the low wavelength region, we also observed variations in the intensities of the modes related to the biochemical groups associated with PCa.\u003c/p\u003e\n\u003cp\u003eAccording to the principal component analysis (PCA) of the spectra, a statistically significant distinction can be observed between the spectra obtained in the control and sarcomatoid and basaloid samples. This statistically substantial distinction indicates differences in the spectral characteristics of these groups. These differences can be explored to develop more accurate and efficient assessment methods for these types of tissues.\u003c/p\u003e\n\u003cp\u003eWhen performing an analysis of the spectral data of the basaloid and sarcomatoid subtypes, statistical significance was found, indicating the differences in the spectral characteristics. The fact that the ellipses are close together suggests a relationship or similarity between the basaloid and sarcomatoid subtypes. This fact means that although there are statistically significant differences in spectral characteristics, overlap or proximity exists in some areas of these subtypes. These results are important because they suggest exploring the similarities and differences identified to develop more accurate and efficient assessment methods to distinguish between control, basaloid, and sarcomatoid groups. In other words, multivariate analysis provides information about the global variation in spectral data. It highlights different and shared aspects between these groups, which can be useful in developing more refined assessment approaches.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eThe study investigated the ultrastructural and vibrational properties of tissue subtypes of penile cancer (PCa) using Atomic Force Microscope (AFM) and Raman Spectroscopy (ER). AFM maps revealed ultrastructural changes in\u0026nbsp;PCa tissues, indicating wear and stretching, possibly due to cytoskeleton and\u0026nbsp;ECM reorganization. Increased roughness of tissues affected by cancer suggests membrane erosion related to tumor invasion. The loss of proteins and lipids in cancer cells may contribute to this roughness, affecting cellular communication and cancer growth and the increased expression of transport proteins, such as ion channels, ion transporters, and aquaporins in metastatic cancer cells.\u003c/p\u003e\n\u003cp\u003eAnalysis of the surface area showed a reduction, especially in the basaloid subtype, associated with aggressiveness and high nodal metastasis. The decrease in basaloid and sarcomatoid sample volumes suggests possible necrosis, lack of blood supply, and cell volume regulation during exposure to osmotic stress. High rates of mitosis and areas of necrosis in these subtypes influence area and volume measurements. Hypoxic conditions in the collagen-rich extracellular matrix, related to factors such as HIF-1, LOX, and Metalloproteinase, affect vascular supply.\u003c/p\u003e\n\u003cp\u003eER detected vibrational modes in PCa tissues, revealing spectral differences between control and cancerous samples. Multivariate analysis confirmed the techniques\u0026apos; discriminatory capacity, indicating significant differences between control and sarcomatoid/basaloid samples. These discoveries could potentially promote precise and efficient physical approaches to cancer, allowing for earlier detection and more targeted treatment. Understanding these characteristics represents a significant advance in oncology.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGMENTS\u003c/strong\u003e: The authors thank CAPES (001), UFMA, HUUFMA, and FAPEMA for supporting this research development.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFUNDING\u003c/strong\u003e: This study was funded by CAPES Financial Code 001, FAPEMA, Projeto Universal-06929/22, Bolsa de Produtividade CNPq, Luciana Magalh\u0026atilde;es Rebelo Alencar - 304774/2021-9.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONFLICTS OF INTEREST\u003c/strong\u003e: All authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eETHICS APPROVAL AND CONSENT TO PARTICIPATE:\u0026nbsp;\u003c/strong\u003eThe study received approval from the Research Ethics Committee on Human Subjects at the University Hospital of the Federal University of Maranh\u0026atilde;o (CEP/HU-UFMA) (CAAE 30760420.3.0000.5086) and was conducted by the Declaration of Helsinki. Considering the study\u0026apos;s retrospective nature, the Research Ethics Committee on Human Subjects at the University Hospital of the Federal University of Maranh\u0026atilde;o exempted the need for patient consent (Opinion Number 4.228.789).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eChristodoulidou M, Sahdev V, Houssein S, Muneer A (2015) Epidemiology of Penile Cancer. Curr Probl Cancer 39:126\u0026ndash;136\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTeixeira J\u0026uacute;nior AAL, da Costa Melo SP, Pinho JD, Sobrinho TBM, Rocha TMS, Duarte DRD, de Oliveira Barbosa L, Duarte WE, de Castro Belfort MR, Duarte KG, da Silva Neto AL, de Calixto RR, Paiva Paiva J, do Nascimento LC, Alencar Junior FSMS, Khayat AM, Lages AS, dos Reis JS, Ara\u0026uacute;jo RB, Silva WS (2022) G.E.B. A Comprehensive Analysis of Penile Cancer in the Region with the Highest Worldwide Incidence Reveals New Insights into the Disease. \u003cem\u003eBMC Cancer\u003c/em\u003e, \u003cem\u003e22\u003c/em\u003e, 1063\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCoelho RWP, Pinho JD, Moreno JS, Garbis DVEO; do, Nascimento AMT, Larges JS, Calixto JRR, Ramalho LNZ, da Silva AAM, Nogueira LR, de Moura Feitoza L, Silva (2018) G.E.B. Penile Cancer in Maranh\u0026atilde;o, Northeast Brazil: The Highest Incidence Globally? \u003cem\u003eBMC Urol\u003c/em\u003e, \u003cem\u003e18\u003c/em\u003e, 50\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSanchez DF et al (2015) HPV- and non-HPV-related subtypes of penile squamous cell carcinoma (SCC): Morphological features and differential diagnosis according to the new WHO classification (2015)., \u003cem\u003eSemin. Diagn. Pathol.\u003c/em\u003e, vol. 32, n\u003csup\u003eo\u003c/sup\u003e 3, pp. 198\u0026ndash;221, maio\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCubilla AL et al (2018) The World Health Organisation 2016 classification of penile carcinomas: a review and update from the International Society of Urological Pathology expert-driven recommendations, \u003cem\u003eHistopathology\u003c/em\u003e, vol. 72, n\u003csup\u003eo\u003c/sup\u003e 6, pp. 893\u0026ndash;904\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCubilla AL, Reuter V, Velazquez E, Piris A, Saito S, Young eRH (2001) Histologic classification of penile carcinoma and its relation to outcome in 61 patients with primary resection., \u003cem\u003eInt. J. Surg. Pathol.\u003c/em\u003e, vol. 9, n\u003csup\u003eo\u003c/sup\u003e 2, pp. 111\u0026ndash;120, abr\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHenry MAC, de Lerco A, Oliveira MM, de Rodrigues WK (2007) Carcinoma basal\u0026oacute;ide escamoso: uma forma rara e agressiva de c\u0026acirc;ncer do es\u0026ocirc;fago e revis\u0026atilde;o da literatura. ABCD arq bras cir dig 20:62\u0026ndash;64\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCubilla AL (2009) The Role of Pathologic Prognostic Factors in Squamous Cell Carcinoma of the Penis. World J Urol 27:169\u0026ndash;177\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOrganization WH, INTERNATIONAL AGENCY FOR RESEARCH ON CANCER IARC Monographs on the (2007) Evaluation of Carcinogenic Risks to Humans: Volume 90 Human Papillomaviruses, vol. 90\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGheit T (2019) Mucosal and Cutaneous Human Papillomavirus Infections and Cancer Biology. Front Oncol 9:355\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWilliams VM, Filippova M, Soto U, Duerksen-Hughes ePJ (2011) HPV-DNA integration and carcinogenesis: putative roles for inflammation and oxidative stress., \u003cem\u003eFuture Virol.\u003c/em\u003e, vol. 6, n\u003csup\u003eo\u003c/sup\u003e 1, pp. 45\u0026ndash;57, jan\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDa MLR, Silva et al (2021) jul., The role of HPV-induced epigenetic changes in cervical carcinogenesis (Review)., \u003cem\u003eBiomed. reports\u003c/em\u003e, vol. 15, n\u003csup\u003eo\u003c/sup\u003e 1, p. 60\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYim E-K, Park eJ-S (2005) The role of HPV E6 and E7 oncoproteins in HPV-associated cervical carcinogenesis., \u003cem\u003eCancer Res. Treat.\u003c/em\u003e, vol. 37, n\u003csup\u003eo\u003c/sup\u003e 6, pp. 319\u0026ndash;324, dez\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSilva DFB, Santos HB, de Le\u0026oacute;n P, Gomes JE, de Alves DQ, Nonaka PM (2019) Clinicopathological and Immunohistochemical Analysis of Spindle Cell Squamous Cell Carcinoma of the Tongue: A Rare Case. Einstein (Sao Paulo) 17:eRC4610\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWHO.Tumours of the penis (2016) In: Who Classification of Tumours of the urinary system and male genital organs. Vol. 4, pp. 259\u0026ndash;285\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEdge SB, Byrd DR, Compton CC, Fritz AG, Greene FL (2009) Trotti. AJCC Cancer Staging Manual, 7th edn. Springer, New York, pp 1\u0026ndash;649\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGravitt PE et al (jan. 2000) Improved amplification of genital human papillomaviruses. J Clin Microbiol 38:357\u0026ndash;361n\u003csup\u003eo\u003c/sup\u003e 1\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDias Rates ER, Almeida CD, de Costa PF, Jansen E, de Farias M, Santos-Oliveira R, Rebelo R, Alencar LM (2024) Evaluation of Biophysical Alterations in the Epithelial and Endothelial Layer of Patients with Bullous Keratopathy. Exp Eye Res 240:109791\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDegiorgio V, Corti M (1985) \u003cem\u003ePhysics of Amphiphiles: Micelles, Vesicles and Microemulsions: Proceedings of the International School of Physics, Enrico Fermi, Course Xc\u003c/em\u003e; Degiorgio, V.; Corti, M., Eds.; 1st ed.; \u0026lrm; Elsevier Science Ltd, ; Vol. 1\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOng YH, Lim M, Liu Q (2012) Comparison of Principal Component Analysis and Biochemical Component Analysis in Raman Spectroscopy for the Discrimination of Apoptosis and Necrosis in K562 Leukemia Cells. Opt Express OE 20:22158\u0026ndash;22171\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePrado-Mantilla A, Lechler T (2023) Polarity in skin development and cancer. Curr Top Dev Biol 154:317\u0026ndash;336\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBaněčkov\u0026aacute; M, Cox D (2023) Top 10 Basaloid Neoplasms of the Sinonasal Tract. Head Neck Pathol 17(1):16\u0026ndash;32\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGoyal S, Rathore R, Sharma S, Arora VK, Das GK, Singal A (2017) Cutaneous basal cell carcinoma with mixed histology: Cytomorphological features of two unusual cases. J Cytol 34(2):115\u0026ndash;118\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRaza MA, Mazzara PF (2011) Sarcomatoid carcinoma of esophagus. Arch Pathol Lab Med 135(7):945\u0026ndash;948\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLosquadro WD (2017) Anatomy of the Skin and the Pathogenesis of Nonmelanoma Skin Cancer, \u003cem\u003eFacial Plast. Surg. Clin. North Am.\u003c/em\u003e, vol. 25, n\u003csup\u003eo\u003c/sup\u003e 3, pp. 283\u0026ndash;289\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHumphrey PA, Moch H, Cubilla AL, Ulbright TM, Reuter eVE (jul. 2016) WHO Classification of Tumours of the Urinary System and Male Genital Organs-Part B: Prostate and Bladder Tumours. Eur Urol 70:106\u0026ndash;119n\u003csup\u003eo\u003c/sup\u003e 1\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRibatti D, Pezzella eF (2021) Overview on the Different Patterns of Tumor Vascularization., \u003cem\u003eCells\u003c/em\u003e, vol. 10, n\u003csup\u003eo\u003c/sup\u003e 3, mar\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang H-S et al (2012) Novel antivirals inhibit early steps in HPV infection., \u003cem\u003eAntiviral Res.\u003c/em\u003e, vol. 93, n\u003csup\u003eo\u003c/sup\u003e 2, pp. 280\u0026ndash;287, fev\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHabiger C, J\u0026auml;ger G, Walter M, Iftner T, Stubenrauch eF (2016) Interferon Kappa Inhibits Human Papillomavirus 31 Transcription by Inducing Sp100 Proteins., \u003cem\u003eJ. Virol.\u003c/em\u003e, vol. 90, n\u003csup\u003eo\u003c/sup\u003e 2, pp. 694\u0026ndash;704, jan\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYan H et al (2019) Efficient Inhibition of Human Papillomavirus Infection by L2 Minor Capsid-Derived Lipopeptide., \u003cem\u003eMBio\u003c/em\u003e, vol. 10, n\u003csup\u003eo\u003c/sup\u003e 4, ago\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMichalczyk K, Misiek M, Chudecka-Głaz eA (2022) Can Adjuvant HPV Vaccination Be Helpful in the Prevention of Persistent/Recurrent Cervical Dysplasia after Surgical Treatment?-A Literature Review., \u003cem\u003eCancers (Basel).\u003c/em\u003e, vol. 14, n\u003csup\u003eo\u003c/sup\u003e 18, set\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTakeishi N, Imai eY (2017) Capture of microparticles by bolus flow of red blood cells in capillaries., \u003cem\u003eSci. Rep.\u003c/em\u003e, vol. 7, n\u003csup\u003eo\u003c/sup\u003e 1, p. 5381, jul\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMurrant e CL, Fletcher NM (2022) Capillary communication: the role of capillaries in sensing the tissue environment, coordinating the microvascular, and controlling blood flow., \u003cem\u003eAm. J. Physiol. Heart Circ. Physiol.\u003c/em\u003e, vol. 323, n\u003csup\u003eo\u003c/sup\u003e 5, pp. H1019\u0026ndash;H1036, nov\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVelazquez EF, Melamed J, Barreto JE, Aguero F, Cubilla AL (2005) Sarcomatoid Carcinoma of the Penis: A Clinicopathologic Study of 15 Cases. Am J Surg Pathol 29:1152\u0026ndash;1158\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAl Tanoury Z, Schaffner-Reckinger E, Halavatyi A, Hoffmann C, Moes M, Hadzic E, Catillon M, Yatskou M, Friederich E (2010) Quantitative Kinetic Study of the Actin-Bundling Protein L-Plastin and of Its Impact on Actin Turn-Over. PLoS ONE 5:e9210\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu S, Xu H, Wang W, Li S, Li H, Li T, Zhang W, Yu X, Liu L (2019) The Role of Collagen in Cancer: From Bench to Bedside. J Translational Med 17:309\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMeira DD, Marinho-Carvalho MM, Teixeira CA, Veiga VF, Da Poian AT, Holandino C, de Freitas MS, Sola-Penna M (2005) Clotrimazole Decreases Human Breast Cancer Cells Viability through Alterations in Cytoskeleton-Associated Glycolytic Enzymes. Mol Genet Metab 84:354\u0026ndash;362\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYim E-K, Park J-S (2005) The Role of HPV E6 and E7 Oncoproteins in HPV-Associated Cervical Carcinogenesis. Cancer Res Treat 37:319\u0026ndash;324\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen J, Weihs D, Van Dijk M, Vermolen eFJ (2018) A phenomenological model for cell and nucleus deformation during cancer metastasis., \u003cem\u003eBiomech. Model. Mechanobiol.\u003c/em\u003e, vol. 17, n\u003csup\u003eo\u003c/sup\u003e 5, pp. 1429\u0026ndash;1450, out\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu W et al (2022) Cancer cell mechanobiology: a new frontier for cancer research, \u003cem\u003eJ. Natl. Cancer Cent.\u003c/em\u003e, vol. 2, n\u003csup\u003eo\u003c/sup\u003e 1, pp. 10\u0026ndash;17\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMadzharova F, Heiner Z, G\u0026uuml;hlke M, Kneipp J (2016) Surface-Enhanced Hyper-Raman Spectra of Adenine, Guanine, Cytosine, Thymine, and Uracil. J Phys Chem C 120:15415\u0026ndash;15423\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMovasaghi Z, Rehman S, Rehman IU (2007) Raman Spectroscopy of Biological Tissues. Appl Spectrosc Rev 42:493\u0026ndash;541\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRygula A, Majzner K, Marzec KM, Kaczor A, Pilarczyk M, Baranska M (2013) Raman Spectroscopy of Proteins: A Review. J Raman Spectrosc 44:1061\u0026ndash;1076\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCzamara K, Majzner K, Pacia MZ, Kochan K, Kaczor A, Baranska M (2015) Raman Spectroscopy of Lipids: A Review. J Raman Spectrosc 46:4\u0026ndash;20\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePezzotti G (2021) Raman Spectroscopy in Cell Biology and Microbiology. J Raman Spectrosc 52:2348\u0026ndash;2443\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDodo K, Fujita K, Sodeoka M (2022) Raman Spectroscopy for Chemical Biology Research. J Am Chem Soc\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOzawa M, Kemler R (1998) Altered Cell Adhesion Activity by Pervanadate Due to the Dissociation of Alpha-Catenin from the E-Cadherin.Catenin Complex. J Biol Chem 273:6166\u0026ndash;6170\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZemła J, Danilkiewicz J, Orzechowska B, Pabijan J, Seweryn S, Lekka M (2018) Atomic Force Microscopy as a Tool for Assessing the Cellular Elasticity and Adhesiveness to Identify Cancer Cells and Tissues. Semin Cell Dev Biol 73:115\u0026ndash;124\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThomas A, Necchi A, Muneer A, Tobias-Machado M, Tran ATH, Van Rompuy A-S, Spiess PE, Albersen M (2021) Penile Cancer. Nat Rev Dis Primers 7:1\u0026ndash;24\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJacob JM et al (2019) mar., Comparative Genomic Profiling of Refractory and Metastatic Penile and Nonpenile Cutaneous Squamous Cell Carcinoma: Implications for Selection of Systemic Therapy., \u003cem\u003eJ. Urol.\u003c/em\u003e, vol. 201, n\u003csup\u003eo\u003c/sup\u003e 3, pp. 541\u0026ndash;548\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang J, Zhang L, Wan D, Zhou L, Zheng S, Lin S, Qiao Y (2021) Extracellular Matrix and Its Therapeutic Potential for Cancer Treatment. Signal Transduct Target Ther 6:153\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMorishita K, Watanabe K, Ichijo H (2019) Cell Volume Regulation in Cancer Cell Migration Driven by Osmotic Water Flow. Cancer Sci 110:2337\u0026ndash;2347\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEre\u0026ntilde;o C, Gaafar A, Garmendia M, Etxezarraga C, Bilbao FJ, L\u0026oacute;pez JI (2008) Basaloid Squamous Cell Carcinoma of the Head and Neck: A Clinicopathological and Follow-up Study of 40 Cases and Review of the Literature. Head Neck Pathol 2:83\u0026ndash;91\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGupta B, Bhattacharyya A, Singh A, Sah K, Gupta V (2018) Basaloid Squamous Cell Carcinoma \u0026ndash; A Rare and Aggressive Variant of Squamous Cell Carcinoma: A Case Report and Review of Literature. Natl J Maxillofac Surg 9:64\u0026ndash;68\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMorishita K, Watanabe K, Ichijo eH (2019) Cell volume regulation in cancer cell migration driven by osmotic water flow. Cancer Sci 110:2337\u0026ndash;2347n\u003csup\u003eo\u003c/sup\u003e 8\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStroka KM, Jiang H, Chen S-H, Tong Z, Wirtz D, Sun SX, Konstantopoulos K (2014) Water Permeat Drives Tumor Cell Migration Confined Microenvironments Cell 157:611\u0026ndash;623\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOster GF, Perelson eAS (1987) The Physics of Cell Motility, \u003cem\u003eJ. Cell Sci.\u003c/em\u003e, vol. n\u003csup\u003eo\u003c/sup\u003e Supplement_8, pp. 35\u0026ndash;54, 1987\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRosengren S, Henson PM, Worthen eGS (1994) Migration-associated volume changes in neutrophils facilitate the migratory process in vitro, \u003cem\u003eAm. J. Physiol. Physiol.\u003c/em\u003e, vol. 267, n\u003csup\u003eo\u003c/sup\u003e 6, pp. C1623\u0026ndash;C1632\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLoitto V-M, Forslund T, Sundqvist T, Magnusson K-E, Gustafsson eM (2002) Neutrophil leukocyte motility requires directed water influx, \u003cem\u003eJ. Leukoc. Biol.\u003c/em\u003e, vol. 71, n\u003csup\u003eo\u003c/sup\u003e 2, pp. 212\u0026ndash;222\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRosengren S, Henson PM, Worthen GS (1994) Migration-Associated Volume Changes in Neutrophils Facilitate the Migratory Process in Vitro. Am J Physiol 267:C1623\u0026ndash;1632\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMehrabi M, Amini F, Mehrabi S (2018) Active Role of the Necrotic Zone in Desensitization of Hypoxic Macrophages and Regulation of CSC-Fate: A Hypothesis. \u003cem\u003eFront. Oncol.\u003c/em\u003e, \u003cem\u003e8\u003c/em\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu Z-G, Jiao D, Necroptosis (2019) Tumor Necrosis and Tumorigenesis. Cell Stress 4:1\u0026ndash;8\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJain RK, Martin JD, Stylianopoulos T (2014) The Role of Mechanical Forces in Tumor Growth and Therapy. Annu Rev Biomed Eng 16:321\u0026ndash;346\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePenna LS, Henriques JAP, Bonatto eD (2017) Anti-mitotic agents: Are they emerging molecules for cancer treatment? \u003cem\u003ePharmacol. Ther.\u003c/em\u003e, vol. 173, pp. 67\u0026ndash;82, maio\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu Y et al (jan. 2024) Developing a prognosis and chemotherapy evaluating model for colon adenocarcinoma based on mitotic catastrophe-related genes. Sci Rep 14:1655n\u003csup\u003eo\u003c/sup\u003e 1\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKriaucionis S, Tahiliani eM (2014) Expanding the epigenetic landscape: novel modifications of cytosine in genomic DNA., \u003cem\u003eCold Spring Harb. Perspect. Biol.\u003c/em\u003e, vol. 6, n\u003csup\u003eo\u003c/sup\u003e 10, p. a018630, out\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOuderkirk JL, Krendel eM (2014) Non-muscle myosins in tumor progression, cancer cell invasion, and metastasis., \u003cem\u003eCytoskeleton (Hoboken).\u003c/em\u003e, vol. 71, n\u003csup\u003eo\u003c/sup\u003e 8, pp. 447\u0026ndash;463, ago\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGebrekiristos M, Melson J, Jiang A, Buckingham eL (jun. 2022) DNA methylation and miRNA expression in colon adenomas compared with matched normal colon mucosa and carcinomas. Int J Exp Pathol 103:74\u0026ndash;82n\u003csup\u003eo\u003c/sup\u003e 3\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMatthews Q, Jirasek A, Lum J, Duan X, Brolo eAG (2010) Variability in Raman spectra of single human tumor cells cultured in vitro: Correlation with cell cycle and culture confluency. Appl Spectrosc 64:871\u0026ndash;887n\u003csup\u003eo\u003c/sup\u003e 8\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Penile Cancer, Biomechanics, Atomic Force Microscopy, Biophysics, Raman Spectroscopy, Tumor","lastPublishedDoi":"10.21203/rs.3.rs-4559053/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4559053/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePenile cancer, although uncommon on a global scale, predominantly arises from squamous cell carcinomas (SCCs). Its occurrence is notably higher in low- and middle-income countries, highlighting the geographic and socioeconomic disparities in the prevalence of this disease. The diversity and significant public health impact of penile cancer underscores the need for new approaches. Nanotechnology, especially through Atomic Force Microscopy (AFM), is promising for studying the nanoscale properties of penile tumor tissues and cells. AFM provides high-resolution topographic images, allowing you to examine the ultrastructural features of cancerous cells and tissues in detail. This helps better understand tumor biomechanics, cell adhesion, morphology, and tumor microenvironment. Raman Spectroscopy (RS) is a powerful technique that detects and analyzes cellular or tissue samples based on morphological characteristics. It scatters photons by molecules polarized by a laser beam, generating a spectral image that reflects the cell's or tissue's chemical composition. This technique can identify changes in the components of cells and tissues, indicative of the presence or progression of the disease. This study proposes to apply RS to investigate the vibrational properties of penile tumor cells and tissues compared with non-tumor counterparts. In this study, through the use of AFM and RS, samples of the subtypes of penile cancer, basaloid and sarcomatoid, as well as non-tumor samples, were analyzed to apply a physical approach to investigate the ultrastructural and vibrational morphology of penile cancer, taking as main tools AFM and RS, providing new information about its nanoscale ultrastructure and offering a new understanding of cancer behavior beyond its molecular composition.\u003c/p\u003e","manuscriptTitle":"Penile Cancer: Innovations in Ultrastructural and Vibrational Markers","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-03 18:53:00","doi":"10.21203/rs.3.rs-4559053/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"329ffb76-72ac-4c06-a010-ef969214f403","owner":[],"postedDate":"July 3rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-08-05T17:40:31+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-03 18:53:00","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4559053","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4559053","identity":"rs-4559053","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}