Deposition of collagen III and alterations in basement membrane integrity as candidate prognostic markers in prostate cancer.

The extracellular matrix surrounding the tumor undergoes changes in its organization during the metastasis process. The present study aims to quantify total collagen, collagen I (Col I) and collagen III (Col III), analyze the alignment of collagen fibers and assess the basement membrane integrity in samples from patients with metastatic and non-metastatic prostate cancer. Tissue samples from 60 patients were classified into groups based on prognostic parameters: better prognosis (n = 20), worse prognosis without metastasis (n = 23) and metastatic (n = 17). Picrosirius red with further analysis under polarizing microscope was used to quantify (with validation using immunohistochemistry) and analyze collagen alignment, and Periodic Acid Schiff staining was used to analyze the basement membrane integrity. The Col I/Col III ratio was found to be higher in the metastatic group than in the groups with better prognosis (p = 0.012) and worse prognosis without metastasis (p = 0.018). Basement membrane integrity constitution in malignant tumor tissue differed from that of adjacent non-tumor tissue (p < 0.001). Moreover, the worsening in the tumor tissue integrity was positively correlated with worse prognostic parameters. All in all, absence of Col III and basement membrane integrity might be indicators of poor prognosis in prostate cancer.

Effects of docetaxel on metastatic prostate (DU-145) carcinoma cells cultured as 2D monolayers and 3D multicellular tumor spheroids.

Docetaxel (DTX) is one of the chemotherapeutic drugs indicated as a first-line treatment against metastatic prostate cancer (mPCa). This study aimed to compare the impact of DTX on mPCa (DU-145) tumor cells cultured as 2D monolayers and 3D multicellular tumor spheroids (MCTS) in vitro. The cells were treated with DTX (1-96 µM) at 24, 48, or 72 hr in cell viability assays (resazurin, phosphatase acid, and lactate dehydrogenase). Cell death was assessed with fluorescent markers and proliferation by clonogenic assay (2D) and morphology, volume, and integrity assay (3D). The cell invasion was determined using transwell (2D) and extracellular matrix (ECM) (3D). Results showed that DTX decreased cell viability in both culture models. In 2D, the IC50 (72 hr) values were 11.06 µM and 14.23 µM for resazurin and phosphatase assays, respectively. In MCTS, the IC50 values for the same assays were 114.9 µM and 163.7 µM, approximately 10-fold higher than in the 2D model. The % of viable cells decreased, while the apoptotic cell number was elevated compared to the control in 2D. In 3D spheroids, only DTX 24 µM induced apoptosis. DTX (≥24 µM at 216 hr) lowered the volume, and DTX 96 µM completely disintegrated the MCTS. DTX reduced the invasion of mPCa cells to matrigel (2D) and migration from MCTS to the ECM. Data demonstrated significant differences in drug response between 2D and 3D cell culture models using mPCa DU-145 tumor cells. MCTS resembles the early stages of solid tumors in vivo and needs to be considered in conjunction with 2D cultures when searching for new therapeutic targets.

Metalloproteinase 9 immunostaining profile is positively correlated with tumor grade, extraprostatic extension and biochemical recurrence in prostate cancer.

Metastasis is the main problem in the treatment of prostate cancer (PCa), and for it to occur, proteolytic enzymes must remodel the extracellular matrix (ECM) surrounding the tumor. The most important group of enzymes with this action include the matrix metalloproteinases (MMPs), which act on various substrates cleaving ECM components. The present study aimed to evaluate the protein immunostaining profiles of matrix metalloproteinase 2 (MMP-2) and 9 (MMP-9) in PCa Brazilian patients using the indirect immunohistochemical methodology. The tissue samples (n = 178), 60 from malignant tumor, 58 from adjacent non-tumor, and 60 from ECM, were evaluated according to the immunostaining intensity. The malignant tumor cytoplasmic MMP-2 immunostaining was more intense than in ECM (p = 0.001), but it did not correlate with any clinical-pathological parameter. The MMP-9 staining was similar in tumor cytoplasm, adjacent non-tumor cytoplasm and ECM, but showed significant positive correlations with ISUP grade (p = 0.044; Tau=0.249), extraprostatic extension (p = 0.025; Tau=0.309), and biochemical recurrence (p = 0.048; Tau=0.306). A significant positive correlation was also observed between MMP-2 and MMP-9 in all cell compartments analyzed. Although further research is warranted to elucidate the precise mechanisms underlying these observations, our findings suggest MMP-9 as a promising candidate marker for tissue invasion that could be used in predicting the progression and prognosis of PCa.

Allelic variants and immunostaining profile in CXCL12/CXCR4 axis: An investigation of association with prognosis in prostate cancer.

Prostate cancer (PCa) is the malignant neoplasm that most commonly affects men and is an important cause of death. It can be detected by changes in serum levels of Prostate Specific Antigen (PSA) and digital rectal examination, but often symptoms do not appear until advanced stages and metastases. The C-X-C Motif Chemokine Ligand 12/C-X-C Motif Chemokine Receptor 4 (CXCL12/CXCR4) axis acts in cell migration and may be involved in the metastatic process. In this context, the aim of this study was to evaluate the allelic variants rs1801157 (CXCL12) and rs2228014 (CXCR4) and the immunostaining of CXCR4 protein as candidates for prognostic markers in PCa. Samples (n = 60) were divided according to prognostic parameters (with and without metastasis at diagnosis) in tree groups: better prognosis, worse prognosis with metastasis at diagnosis and worse prognosis without metastasis at diagnosis, and immunostaining was evaluated by indirect immunohistochemistry, considering tumoral and adjacent tissues from the same patient (n = 120). A significant association was found between the C allele of rs2228014 (CXCR4) and the extraprostatic extension. For CXCR4 immunostaining a weak labeling and a cytoplasmic localization predominated, as well as a significant difference between malignant versus adjacent tissue, with higher protein expression in the malignant tissue. A significant association was found between CXCR4 tumor immunostaining with TNM staging (T2b-T2c) and PSA level (> 20 ng/mL). None of the allelic variants affected CXCR4 immunostaining. Prognostic groups did not differ in allelic variant frequency or immunostaining profile. Findings suggest that CXCR4 receptor may be one of the ways to worsen the prognosis of prostatic cancer.

Three-dimensional cell cultures as preclinical models to assess the biological activity of phytochemicals in breast cancer.

The demand for the development of three-dimensional (3D) cell culture models in both/either drug screening and/or toxicology is gradually magnified. Natural Products derived from plants are known as phytochemicals and serve as resources for novel drugs and cancer therapy. Typical examples include taxol analogs (i.e., paclitaxel and docetaxel), vinca alkaloids (i.e., vincristine, vinblastine), and camptothecin analogs (topotecan, irinotecan). Breast cancer is the most frequent malignancy in women, with a 70% chance of patients being cured; however, metastatic disease is not considered curable using currently available chemotherapeutic options. In addition, phytochemicals present promising options for overcoming chemotherapy-related problems, such as drug resistance and toxic effects on non-target tissues. In the toxicological evaluation of these natural compounds, 3D cell culture models are a powerful tool for studying their effects on different tissues and organs in similar environments and behave as if they are in vivo conditions. Considering that 3D cell cultures represent a valuable platform for identifying the biological features of tumor cells as well as for screening natural products with antitumoral activity, the present review aims to summarize the most common 3D cell culture methods, focusing on multicellular tumor spheroids (MCTS) of breast cancer cell lines used in the discovery of phytochemicals with anticancer properties in the last ten years.

Tissue immunostaining of candidate prognostic proteins in metastatic and non-metastatic prostate cancer.

PURPOSE: Prostate cancer (PCa) lacks specific markers capable of distinguishing aggressive tumors from those with indolent behavior. Therefore, the aim of this study was to evaluate the immunostaining of candidate proteins (PTEN, AKT, TRPM8, and NKX3.1) through the immunohistochemistry technique (IHC) on patients with metastatic and non-metastatic PCa. METHODS: Tissues from 60 patients were divided into three groups categorized according to prognostic parameters: better prognosis (n = 20), worse prognosis (n = 23), and metastatic (n = 17). Immunostaining was analyzed by a pathologist and staining classifications were considered according to signal intensity: (0) no staining, (+) weak, and (++ and +++) intermediate to strong. RESULTS: AKT protein was associated (p = 0.012) and correlated (p = 0.014; Tau = - 0.288) with the prognostic groups. The immunostaining for TRPM8 (p = 0.010) and NKX3.1 (p = 0.003) proteins differed between malignant tumor and non-tumoral adjacent tissue as well as for proteins in cellular locations (nucleus and cytoplasm). TRPM8 was independently associated with the ISUP grade ≥ 4 (p = 0.024; OR = 8.373; 95% CI = 1.319-53.164). The NKX3.1 showed positive and predominantly strong immunostaining in all patients in both tumoral and non-tumoral adjacent tissues. All metastatic samples had positive immunostaining, with strong intensity for NKX3.1 (p = 0.021; Tau = - 0.302). In the non-metastatic group, this strong protein staining was not observed in any patients. CONCLUSION: This study confirmed that NKX3.1 is highly specific for prostate tissue and indicated that NKX3.1, AKT, and TRPM8 may be candidate markers for prostate cancer prognosis.

The potential of cell-free and exosomal microRNAs as biomarkers in liquid biopsy in patients with prostate cancer.

PURPOSE: Prostate cancer (PCa) is the 4th most diagnosed cancer and the 8th leading cause of cancer-related death worldwide. Currently, clinical risk stratification models including factors like PSA levels, Gleason score, and digital rectal examination are used for this purpose. There is a need for novel biomarkers that can distinguish between indolent and aggressive pathology and reduce the risk of overdiagnosis/overtreatment. Liquid biopsy has a non-invasive character, can lead to less morbidity and provide new biomarkers, such as miRNAs, that regulate diverse important cellular processes. Here, we report an extended revision about the role of cell-free and exosomal miRNAs (exomiRNAs) as biomarkers for screening, diagnosis, prognosis, or treatment of PCa. METHODS: A comprehensive review of the published literature was conducted focusing on the usefulness, advantages, and clinical applications of cell-free and exomiRNAs in serum and plasma. Using PubMed database 53 articles published between 2012 and 2021 were selected and discussed from the perspective of their use as diagnostic, prognostic and therapeutic biomarkers for PCa. RESULTS: We identify 119 miRNAs associated with PCa development and the cell-free and exosomal miR-21, miR-141, miR-200c, and miR-375 were consistently associated with progression in multiple cohorts/studies. However, standardized experimental procedures, and well-defined and clinically relevant cohort studies are urgently needed to confirm the biomarker potential of cell-free and exomiRNAs in serum or plasma. CONCLUSION: Cell-free and exomiRNAs in serum or plasma are promising tools for be used as non-invasive biomarkers for diagnostic, prognosis, therapy improvement and clinical outcome prediction in PCa patients.

The role of NFKB1/NFKBIA genetic variants in HPV infection: A cross-sectional cohort study.

Human Papillomavirus (HPV) is the most frequent etiological agent sexually transmitted. In the context of the immune response, NF-kB pathway plays an important role controlling the expression of several genes essential to cellular activity and structural and/or functional changes in components of this pathway can promote the development of several tumors. Thus, the study purpose was to evaluate the influence of NFKB1 rs28362491 and NFKBIA rs696 genetic variants on HPV infection and cervical lesions development. In this study 334 patients were recruited, of whom 48.8% (n = 163) were HPV infected, and considered our case group. HPV-DNA was detected by polymerase chain reaction (PCR) and the genetic variants were assessed in blood cells and tumor tissues paraffin embedded samples through restriction fragment length polymorphism analysis. Among women who were recruited for this study who were infected, 37.4% presented precursor lesions and 16.8% were diagnosed with cervical cancer (CC). The present study did not observe significant effects of the interaction between such genetic variants on HPV infection, nor on the development of lesions and progression to CC. Further studies will be important to investigate if under some circumstance the NFKB1 rs28362491 and NFKBIA rs696 genetic variants influence the progression of HPV-associated lesions.

Haplotype Structures and Protein Levels of TGFB1 in HPV Infection and Cervical Lesion: A Case-Control Study.

This study aimed to verify the role of TGFB1 variants (c.-1638G>A, c.-1347C>T, c.29C>T, and c.74G>C) in HPV infection susceptibility and cervical lesions development, and their impact on TGFB1 cervical and plasma levels. TGFB1 genotypes were assessed with PCR-RFLP and haplotypes were inferred for 190 HPV-uninfected and 161 HPV-infected women. TGFB1 levels were determined with immunofluorimetric assay. Case-control analyses were performed with logistic regression adjusted for possible confounders. Women carrying -1347TT or -1347CT+TT as well as those with 29CT, 29CC, or 29CT+CC were more likely to have HPV than -1347CC and 29TT carriers, respectively. Regarding haplotypes, the most frequent were *4 (GCTG) and *3 (GTCG). Women *4/*4 were less likely to have HPV than those with no *4 copy. Comparing the inheritance of *3 and *4, carriers of *3/*4 or *3/*3 were more susceptible to HPV than *4/*4. The TGFB1 plasma and cervical levels were higher in the infected patients. Plasma levels were also higher in infected women with low-grade lesions. HPV-infected patients carrying *3/Other and *3/Other+*3/*3 presented lower TGFB1 plasma levels than those with no copy of *3. TGFB1 variants could contribute to the comprehension of the TGFB1 role in HPV-caused cervical disease.

FOXP3 immunoregulatory gene variants are independent predictors of human papillomavirus infection and cervical cancer precursor lesions.

PURPOSE: FOXP3 is a marker of the T regulatory (Treg) cell subset and drives its function and homeostasis. Its expression maintains the host immunosuppressive state that favors persistence of human papillomavirus (HPV) infection and squamous intraepithelial lesion (SIL) appearance. The present study evaluated the effects of the rs3761548 and rs2232365 intronic single-nucleotide variants (SNVs) and their haplotypes on HPV infection and SIL diagnosis in HPV-infected and -uninfected women. METHODS: HPV DNA-based detection in cervical specimens was performed by PCR. FOXP3 variants were genotyped by PCR-restriction fragment length polymorphism and haplotype recombination sites were inferred for 208 HPV-infected and 218 HPV-uninfected women diagnosed or not with low- or high-grade intraepithelial lesions of cervix. Case-control analyses were carried out by logistic regression adjusted for several socio-demographic, sexual lifestyle, and clinical data. RESULTS: The homozygous genotype of the rs3761548 variants (A/A) (related to decreased FOXP3 expression) may exert a protective role against HPV infection in women (ORAj: 0.60; 95% CI 0.36-0.99; p = 0.049) and was an independent predictor of protection against HSIL development (ORAdj: 0.28; 95% CI 0.11-0.68; p = 0.006). In addition, the homozygous genotype (G/G) of the rs2232365 variants (related to increased FOXP3 expression) was independently associated with the HPV infection (ORAdj: 2.10; 95% CI 1.06-4.15; p = 0.033). Haplotype analysis revealed no significant associations in our study. CONCLUSIONS: Our results reveal the significant and independent associations between FOXP3 genetic variants and susceptibility to HPV infection and SIL diagnosis and their role as biomarkers of HPV infection and cervical lesion management.

Genetic variant in CXCL12 gene raises susceptibility to HPV infection and squamous intraepithelial lesions development: a case-control study.

BACKGROUND: Human papillomavirus (HPV) is the most common sexually transmitted virus in women worldwide. The persistence of the virus may cause warts that are considered benign lesions and low or high grade intraepithelial lesions (LSIL/HSIL). Immunological system plays an important role in the resolution of infections. In this context, we highlight the chemokines, which are important regulators in the development of viral infections and inflammation. Among which CXCL12 stands out, due to its pro-inflammatory features, acting as chemoattractant recruiting immune cells. Several polymorphisms were identified in CXCL12 gene including rs1801157 in the 3'-untranslated region, which is characterized by a substitution of a guanine for an adenine. METHODS: In this study, 195 women were classified as HPV non-infected and 169 as HPV-infected. HPV-DNA was detected by polymerase chain reaction (PCR) and the polymorphism was assessed in blood cells through restriction fragment length polymorphism analysis. RESULTS: HPV infection was more incident in women who had more than 4 sexual partners during lifetime (p = 0.007), among those who presented lower number of pregnancies (p = 0.017). HPV was more prevalent among allele A carriers confirmed by logistic regression analysis adjusted for several confounding factors [ORADJ = 4.985; CI95% (2.85-8.72), p < 0.001]. An association between allele A carriers and HSIL development (p = 0.003) was also observed. CONCLUSIONS: In the present study, we demonstrated that CXCL12 rs1801157 is independently associated with HPV infection and exerts influence in HSIL development, suggesting it as a promising susceptibility biomarker for HPV infection and lesions development.