Tumor Invasive Front Research Articles

Effects of imipramine on cancer patients over-expressing Fascin1; description of the HITCLIF clinical trial.

Tumor invasion and metastasis are responsible for the majority of cancer-related deaths. The identification of molecules involved in these processes is crucial to design effective treatments that can halt the progression of cancer. To spread and metastasize, tumor cells must restructure their cytoskeleton and emit protrusions. A key molecule in this process of creating these invading structures is Fascin1, the main protein involved in the formation of actin cytoskeleton bundles and a consistent marker of bad prognosis in several types of cancer. Recent studies have shown that imipramine, an FDA- and EMA-approved antidepressant, can block Fascin1and prevent the formation of actin bundles, making it a promising candidate for the treatment of Fascin1-expressing cancers. As a result, a clinical trial will be conducted to assess the efficacy of imipramine being the first experimental clinical study selecting patients based on Fascin1 expression. The HITCLIF trial is a multicenter, double-blind, placebo-controlled, randomized and non-commercial phase II clinical trial conducted in parallel groups to evaluate the effectiveness of the tricyclic antidepressant imipramine as anti-invasive agent in the treatment of localized colon, rectal and triple negative breast cancer patients with overexpression of Fascin1. Eligible patients will be randomly assigned, in a 1:1 ratio, to receive imipramine or placebo. Patients will be stratified into 2 groups according to whether administration of imipramine is concomitant with neoadjuvant chemotherapy regimen. Group A will receive imipramine alone without neoadjuvant chemotherapy, while Group B will receive imipramine treatment along with the standard neoadjuvant chemotherapy regimen. The primary endpoint of the trial is the grade of alteration in the prognostic histopathological features at invasive margins (tumor budding, cytoplasmic pseudo-fragments, tumor growth pattern, and peritumoral lymphocytic infiltration). Fascin1 is an interesting therapeutical target as it plays a causative role in the invasion and metastasis of cancer cells. Moreover, its expression is virtually absent in normal epithelia but highly expressed in cancer with bad prognosis. In silico, in vitro and in vivo studies by our group have demonstrated that the antidepressant imipramine has Fascin1-dependant anti-invasive and anti-metastatic effects in colorectal cancer cells. Now we are recruiting patients in a clinical trial based on Fascin1 over-expression in which administration of imipramine will be carried out during the period between the diagnosis biopsy and surgical resection to explore the drug effects on tumor invasive front. https:///www.clinicaltrialsregister.eu/ctr-search/trial/2021-001328-17/ES, identifier 2021-001328-17.

GLDC promotes colorectal cancer metastasis through epithelial-mesenchymal transition mediated by Hippo signaling pathway.

Cancer metastasis remains a major cause of death in cancer patients, and epithelial-mesenchymal transition (EMT) plays a decisive role in cancer metastasis. Recently, abnormal expression of Glycine Decarboxylase (GLDC) has been demonstrated in tumor progression, and GLDC is up-regulated in cancers, such as lung, prostate, bladder, and cervical cancers. However, the exact role of GLDC in colorectal cancer (CRC) progression remains to be elucidated. The aim of our study was to explore the role of GLDC in CRC metastasis. The GSE75117 database was used to investigate GLDC expression in tumor center and invasive front tissues and we found that GLDC expression levels were higher in theinvasive front tissue. GLDC expression levels were negatively correlated with the prognosis of CRC patients. In vitro studies have showed that GLDC can promote invasion and migration of CRC cells by inhibiting the Hippo signaling pathway and regulating the EMT process. Blocking the Hippo signaling pathway with Verteporfin reduced the effect of GLDC on CRC metastasis. In vivo metastasis assays further confirmed that tail vein injection of GLDC+/+ cells induced morelung metastasis, compared to normal CRC cells. The results of this study suggest that GLDC promotes EMT through the Hippo signaling pathway, providing a new therapeutic target for CRC metastasis.

Vascular phenotyping of the invasive front in breast cancer using a 3D angiogenesis atlas.

Vascular remodeling at the invasive tumor front (ITF) plays a critical role in progression and metastasis of triple negative breast cancer (TNBC). Therefore, there is a crucial need to characterize the vascular phenotype (i.e. changes in the structure and function of vasculature) of the ITF and tumor core (TC) in TNBC. This requires high-resolution, 3D structural and functional microvascular data that spans the ITF and TC (i.e. ∼4-5mm from the tumor's edge). Since such data are often challenging to obtain with most conventional imaging approaches, we employed a unique "3D whole-tumor angiogenesis atlas" derived from orthotopic xenografts to characterize the vascular phenotype of the ITF and TC in TNBC. First, high-resolution (8μm) computed tomography (CT) images of "whole-tumor" microvasculature were acquired from eight orthotopic TNBC xenografts, of which three tumors were excised at post-inoculation day 21 (i.e. early-stage) and five tumors were excised at post-inoculation day 35 (i.e. advanced-stage). These 3D morphological CT data were combined with soft tissue contrast from MRI as well as functional data generated in silico using image-based hemodynamic modeling to generate a multi-layered "angiogenesis atlas". Employing this atlas, blood vessels were first spatially stratified within the ITF (i.e. ≤1mm from the tumor's edge) and TC (i.e. >1mm from the tumor's edge) of each tumor xenograft. Then, a novel method was developed to visualize and characterize microvascular remodeling and perfusion changes in terms of distance from the tumor's edge. The angiogenesis atlas enabled the 3D visualization of changes in tumor vessel growth patterns, morphology and perfusion within the ITF and TC. Early and advanced stage tumors demonstrated significant differences in terms of their edge-to-center distributions for vascular surface area density, vascular length density, intervessel distance and simulated perfusion density (p≪0.01). Elevated vascular length density, vascular surface area density and perfusion density along the circumference of the ITF was suggestive of a preferential spatial pattern of angiogenic growth in this tumor cohort. Finally, we demonstrated the feasibility of differentiating the vascular phenotypes of ITF and TC in these TNBC xenografts. The combination of a 3D angiogenesis atlas and image-based hemodynamic modeling heralds a new approach for characterizing the role of vascular remodeling in cancer and other diseases.

Prognostic Nomogram for Lymph-Node Metastasis in Oral Squamous Cell Carcinoma (OSCC) Using Immunohistochemical Marker D2-40.

Nomograms are proven in "individualized risk prediction" in sarcomas and breast and prostate cancers. Incorporating immunohistochemical markers and histopathological parameters can enhance accuracy of these graphical representations of statistical predictive models concerning metastasis. D2-40, a monoclonal antibody to podoplanin (regulator of motility expressed in malignant epithelial cells), dually predicts metastatic potential of tumour by estimating the motile tumour phenotype and by detecting lymphatic vessels/density, both essential to metastasis in OSCC. Thus, we propose a model that incorporates D2-40 immunostaining of individual tumour cells (ITC) too with other variables (seen in H+E staining) as a predictive nomogram. Sixty cases of OSCC were selected with equal number of cases (n=30) of pN0 and pN+ status. Bryne's grading of invasive front of tumour (ITF) was done on H+E-stained slides followed by D2-40 immunostaining for ITCs at ITF and lymphatic vessels. Multivariate regression analysis was used to generate the nomogram of LNM where the predictive contribution of each covariate, namely depth of invasion, D2-40-stained ITCs, gender, histological grade, and worst pattern of invasion (WPOI), was plotted on a scale of 1-100 points. The nomogram showed that the strongest variable in OSCC was the WPOI in H+E-stained section followed by D2-40-positive ITCs and gender. Our predictive nomogram for LNM in OSCC surprisingly showed that a tumour with lower score of WPOI (islands vs ITC) showed numerous D2-40-positive ITCs, drastically increasing the probability of metastasis. The concept of "individualized risk prediction" can be used to predict lymph node metastasis using a variety of histopathological criteria that can be visualized in routine and immunohistochemical staining in OSCC with the aid of a nomogram.

Apoptosis-related Prognostic Factors in Advanced Colorectal Cancer Determined Using Tissue Microarrays.

Cancer cells evade apoptosis in colorectal cancer (CRC); however, overlap between apoptosis and poor prognosis marker proteins in the invasive front of tumors has not been reported. Here, we aimed to clarify the relationship between apoptosis, apoptosis-related protein expression, and prognosis in the central and invasive front regions of CRC using tissue microarrays. Data of 207 patients with pathological stage 3 CRC, who underwent radical surgery between October 2010 and November 2014, were retrospectively reviewed. We assessed apoptosis using M30 CytoDEATH, CD163, and p53 immunostaining in tumor sections in the center and invasive front using tissue microarrays and correlated the results with the survival outcomes. M30 CytoDEATH staining was negative; 134 cases (64.7%) were apoptosis-negative in the center and 103 (49.8%) were apoptosis-negative at the invasive front. CD163 positivity was observed in 16 cases (7.8%) in the center and in 36 cases (17.6%) at the invasive front; p53 positivity was observed in 33 (15.9%) and 64 (30.9%) cases in the center and invasive front, respectively. CD163 and p53 expression was not associated with survival outcomes; however, the apoptosis-negative group at the invasive front had significantly poorer survival outcomes (overall survival: p=0.044, relapse-free survival: p=0.001). We identified cases with a poor prognosis by combining apoptosis and CD163 expression. A lower apoptosis percentage at the invasive front is associated with a poorer prognosis. CRC cases with a poor prognosis can be identified by evaluating apoptosis and CD163 expression in the invasive front.

Single-cell RNA reveals a tumorigenic microenvironment in the interface zone of human breast tumors

BackgroundThe interface zone, area around invasive carcinoma, can be thought of as the actual tissue of the tumor microenvironment with precedent alterations for tumor invasion. However, the heterogeneity and characteristics of the microenvironment in the interface area have not yet been thoroughly explored.MethodsFor in vitro studies, single-cell RNA sequencing (scRNA-seq) was used to characterize the cells from the tumor zone, the normal zone and the interface zone with 5-mm-wide belts between the tumor invasion front and the normal zone. Through scRNA-seq data analysis, we compared the cell types and their transcriptional characteristics in the different zones. Pseudotime, cell–cell communication and pathway analysis were performed to characterize the zone-specific microenvironment. Cell proliferation, wound healing and clone formation experiments explored the function of differentially expressed gene BMPR1B, which were confirmed by tumor models in vivo.ResultsAfter screening, 88,548 high-quality cells were obtained and identified. Regulatory T cells, M2 macrophages, angiogenesis-related mast cells, stem cells with weak DNA repair ability, endothelial cells with angiogenic activity, fibroblasts with collagen synthesis and epithelial cells with proliferative activity form a unique tumorigenic microenvironment in the interface zone. Cell–cell communication analysis revealed that there are special ligand–receptor pairs between different cell types in the interface zone, which protects endothelial cell apoptosis and promotes epithelial cell proliferation and migration, compared to the normal zone. Compared with the normal zone, the highly expressed BMPR1B gene promotes the tumorigenic ability of cancer cells in the interface zone.ConclusionsOur work identified a unique tumorigenic microenvironment of the interface zone and allowed for deeper insights into the tumor microenvironment of breast cancer that will serve as a helpful resource for advancing breast cancer diagnosis and therapy.

Multi-Faceted Role of Cancer-Associated Adipocytes in Colorectal Cancer.

Colorectal cancer (CRC) is one of the most commonly diagnosed types of cancer, especially in obese patients, and the second cause of cancer-related death worldwide. Based on these data, extensive research has been performed over the last decades to decipher the pivotal role of the tumor microenvironment (TME) and its cellular and molecular components in CRC development and progression. In this regard, substantial progress has been made in the identification of cancer-associated adipocytes' (CAAs) characteristics, considering their active role in the CCR tumor niche, by releasing a panel of metabolites, growth factors, and inflammatory adipokines, which assist the cancer cells' development. Disposed in the tumor invasion front, CAAs exhibit a fibroblastic-like phenotype and establish a bidirectional molecular dialogue with colorectal tumor cells, which leads to functional changes in both cell types and contributes to tumor progression. CAAs also modulate the antitumor immune cells' response and promote metabolic reprogramming and chemotherapeutic resistance in colon cancer cells. This review aims to report recent cumulative data regarding the molecular mechanisms of CAAs' differentiation and their activity spectrum in the TME of CRC. A better understanding of CAAs and the molecular interplay between CAAs and tumor cells will provide insights into tumor biology and may open the perspective of new therapeutic opportunities in CRC patients.

Evaluation of Invasive Tumour Front in Primary and Secondary Oral Squamous Cell Carcinoma – A Reliable Prognostic Parameter

Introduction: Oral squamous cell carcinoma (OSCC) is the most common malignant epithelial neoplasm affecting the oral cavity. Recent work indicates that morphological and molecular characteristics of tumour cells at invasive tumor fronts (ITF) underlie the biological aggressiveness of OSCC. The aim of the study was to compare the pattern of invasion (POI) (Type I to V) at tumour–host interface in 150 primary and 50 secondary OSCC cases. Materials and Methods: Both pre-dominant POI and worst POI was evaluated in all cases. Pre-dominant pattern of invasion and worst pattern of invasion (WPOI) were compared with Broder's grading system. Statistical analysis was done using a statistical software package (SPSS) and Fisher's exact test. Results: Results showed that the WPOI pattern also moves towards higher scores as the grade increases. Conclusion: We thus conclude that POI could serve as an individual prognostic marker irrespective of the histologic differentiation of the tumour.

Clinical significance of the expression of FOXP3 and TIGIT in Merkel cell carcinoma

The pathogenesis of 80% of Merkel cell carcinoma (MCC) cases is associated with Merkel cell polyomavirus (MCPyV). Forkhead helix transcription factor P3 (FOXP3) and the T cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibition motif domains (TIGIT)–CD155 pathway, which are targets for immunotherapy, were assessed as prognostic factors of MCC. We analyzed mRNA expression data of 111 patients with MCC and performed immunohistochemical analysis to detect the expression of programmed death ligand 1 (PD-L1), CD8, FOXP3, TIGIT, and CD155 in 65 cases of MCC. In CD8 and FOXP3 immunostaining, the number of expressing-infiltrating cells was determined by dividing the region into tumor center and invasive front areas. FOXP3 expression was evaluated separately in cells with high and low intensities. Aberrant TIGIT expression and weak CD155 staining were observed in MCC cells. CD8- and FOXP3-positive cell infiltrations were higher in the invasive front than in the tumor center. Multivariate Cox hazard analysis revealed that high infiltration of cells with low-intensity FOXP3 expression in the invasive front is a favorable prognostic factor (p = 0.025). Thus, targeting TIGIT–CD155 signaling and FOXP3 as well as PD-L1 may be a therapeutic strategy for MCC.

A novel pathological finding, "BABA", for a prognostic factor in oral tongue carcinoma.

92 Background: Oral tongue carcinoma is one of the cancers with poor prognosis. The UICC staging classification is commonly used to predict outcomes and to select the treatments, although there are cases where cancer relapse early despite being at the same Stages, indicating a need for additional prognostic factors to predict patient outcomes accurately. Recent advances in RNA sequencing of tumor and immune cells, as well as elucidation of the tumor microenvironment, have enabled the identification of many genes associated with recurrence risk and immunosuppressive environment around the tumor, which essential for precision treatment of the patients with high risks of recurrence. However pathological findings remain the first step in selecting samples with a high risk of recurrence for further analysis of RNA sequencing or the tumor microenvironment. In this study, we identified a novel pathological feature named as “BABA” (Broadening Amoeboid Behavior of Advanced tumor cells) that appears around the invasive front of tumors. It describes the way in which cancer cells progress like amoeba between muscle fibers and affect the prognosis of oral tongue carcinoma, making it a potential factor for the future analysis of next-generation sequencing or tumor microenvironment. Methods: Patients with oral tongue squamous cell carcinoma who underwent surgery were recruited, excluding those who had received salvage treatment for recurrence, those with simultaneous occurrence of carcinoma, those who had undergone palliative therapy, postoperative therapy, or those who lacked any pathological findings. Survival analyses for existing pathological findings, such as pT (pathological primary stage), pN (pathological nodal stage), ENE (extra nodal extension) status, lymphatic invasion, venous invasion, perineural extension, margin status, WPOI (worst pattern of invasion) and BABA were conducted. Results: A total number of 211 patients were analyzed in this study. Patients with positive for BABA had a two-year overall survival rate and recurrence-free survival rate of 0.78(p=0.02) and 0.39 (p<0.001), respectively, according to Kaplan-Meier analysis. In the multivariate analysis for overall survival using Cox regression model, venous invasion and pN ≥1 were significant prognostic factors, with HR (95%CI) of 3.0 (1.5-6.3) and 2.4 (1.2-4.5), respectively. In the multivariate analysis for recurrence free survival using Cox regression model, positive for lymphatic invasion, venous invasion, BABA, pN ≥1 were significant factors, with HR (95%CI) of 2.1 (1.2-3.6), 2.0 (1.1-3.4), 2.2 (1.3-3.7) and 2.0 (1.2-3.6), respectively. Conclusions: BABA is the novel risk factor for recurrence in oral tongue carcinoma. Identifying BABA could help clinicians better predict recurrence and develop more personalized treatment strategies.

Pseudobudding: ruptured glands do not represent true tumor buds.

Tumor budding (TB) is a strong biomarker of poor prognosis in colorectal cancer and other solid cancers. TB is defined as isolated single cancer cells or clusters of up to four cancer cells at the invasive tumor front. In areas with a large inflammatory response at the invasive front, single cells and cell clusters surrounding fragmented glands are observed appearing like TB. Occurrence of these small groups is referred to as pseudobudding (PsB), which arises due to external influences such as inflammation and glandular disruption. Using a combination of orthogonal approaches, we show that there are clear biological differences between TB and PsB. TB is representative of active invasion by presenting features of epithelial-mesenchymal transition and exhibiting increased deposition of extracellular matrix within the surrounding tumor microenvironment (TME), whereas PsB represents a reactive response to heavy inflammation where increased levels of granulocytes within the surrounding TME are observed. Our study provides evidence that areas with a strong inflammatory reaction should be avoided in the routine diagnostic assessment of TB. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Protocol for evaluation and correlation of CD44 immunoexpression in tumor tissue and surgical margins of oral squamous cell carcinoma with three-year survival: A retrospective study

Introduction: The commonest type of cancer in the head and neck region is oral squamous cell carcinoma (OSCC) due to its high rates of occurrence and mortality. Cluster of differentiation 44 (CD44) is a surface glycoprotein present on tumor cells. It regulates cell proliferation, adhesion, migration an invasion of cancer stem cell. Immunoexpression of CD44 in surgical margins indicates poor prognosis of disease and increased risk of local recurrence.[4] Objectives: To evaluate and correlate CD44 immunoexpression in tumor tissue and surgical margins OSCC with three-year survival. Methodology: All tissue sections will be processed for CD44 immunohistochemistry. After that, light microscopic evaluation of CD44 expression will be done. A dark brown color of membranous staining of neoplastic cells will be considered for CD44 positivity; this must be observed in the tumor core, in the basal layer because most stem cells are present in the basal layer of oral mucosa and in the invasive front of tumor. Expected results: The present study will find the immunohistochemical expression of CD44 tumor tissue and surgical margins of OSCC, in order to evaluate the prognosis of OSCC. Immunoexpression may vary in according to their different grades. Conclusions: We hypothesize that, as the disease stage advances, intensity of CD44 expression immunohistochemically increases and it can adversely affect overall survival; hence it is considered as an independent predictor of prognosis of OSCC.

Prognostic Impact of the Immune-Cell Infiltrate in N1-Positive Non–Small-Cell Lung Cancer

IntroductionThe tumoral immune milieu plays a crucial role for the development of non–small-cell lung cancer (NSCLC) and may influence individual prognosis. We analyzed the predictive role of immune cell infiltrates after curative lung cancer surgery. Materials and MethodsThe tumoral immune-cell infiltrate from 174 patients with pN1 NSCLC and adjuvant chemotherapy was characterized using immunofluorescence staining. The density and distribution of specific immune cells in tumor center (TU), invasive front (IF) and normal tissue (NORM) were correlated with clinical parameters and survival data. ResultsTumor specific survival (TSS) of all patients was 69.9% at 5 years. The density of tumor infiltrating lymphocytes (TIL) was higher in TU and IF than in NORM. High TIL density in TU (low vs. high: 62.0% vs. 86.7%; p = .011) and the presence of cytotoxic T-Lymphocytes (CTLs) in TU and IF were associated with improved TSS (positive vs. negative: 90.6% vs. 64.7% p = .024). High TIL-density correlated with programmed death-ligand 1 expression levels ≥50% (p < .001). Multivariate analysis identified accumulation of TIL (p = .016) and low Treg density (p = .003) in TU as negative prognostic predictors in squamous cell carcinoma (p = .025), whereas M1-like tumor- associated macrophages (p = .019) and high programmed death-ligand 1 status (p = .038) were associated with better survival in adenocarcinoma. ConclusionThe assessment of specific intratumoral immune cells may serve as a prognostic predictor in pN1 NSCLC. However differences were observed related to adenocarcinoma or squamous cell carcinoma histology. Prospective assessment of the immune-cell infiltrate and further clarification of its prognostic relevance could assist patient selection for upcoming perioperative immunotherapies.

Poor Prognosis in Patients With Stage III Colorectal Cancer Receiving Adjuvant FOLFOX/CAPOX Therapy Is Predicted by the Presence of Many Poorly Differentiated Clusters.

Poorly differentiated clusters (PDCs) have been reported to be a useful grading system for predicting prognosis in patients with colorectal cancer (CRC). We investigated the association between the number of PDCs and prognosis in patients with stage III CRC treated with oxaliplatin-based adjuvant chemotherapy. This is a retrospective study of 49 patients with stage III CRC who underwent curative surgery followed by oxaliplatin-based adjuvant chemotherapy. PDC was defined as a cluster of ≥5 cancer cells without glandular structure at the invasive front of the primary tumor. During the observation period, 12 patients experienced relapse. The patients were divided into two groups (<7 and ≥7 PDC groups), and receiver operating characteristic (ROC) curves were calculated [area under the curve (AUC)=0.743]. Patients with ≥7 PDCs had a much shorter relapse-free survival (RFS) than those with <7 PDCs (p<0.0001). The overall survival (OS) was also significantly worse in patients with ≥7 PDCs than in those with <7 PDCs (p<0.0001). Multivariate analysis revealed that PDC was the only significant prognostic factor measured that could predict RFS (p=0.002) and OS (p=0.0047) in patients with stage III CRC treated with oxaliplatin-based adjuvant chemotherapy. In patients with stage III CRC treated with post-resection oxaliplatin-based adjuvant chemotherapeutic regimens, the presence of ≥7 PDCs at the invasive front of the primary tumor predicted unfavorable prognosis.

E-Cadherin Expression Varies Depending on the Location within the Primary Tumor and Is Higher in Colorectal Cancer with Lymphoid Follicles.

Reliable indicators of cancer advancement have actively been sought recently. The detection of colorectal cancer progression markers is essential in improving diagnostic and therapeutic protocols. The aim of the study was to investigate the profile of E-cadherin expression in colorectal cancer tissue depending on the TNM staging and its correlation with several clinical and histopathological features. The study included 55 colorectal cancer patients admitted to the surgical ward for elective surgery. Tissue samples were obtained from resected specimens. Different distributions of E-cadherin expression within tumors were observed; the highest percentage of positive E-cadherin expression was found in the invasive front and in the tumor center. Additionally, the different cellular distribution of E-cadherin expression was noticed; weak membranous E-cadherin expression was the highest in the invasive front and in the budding sites, but a strong membranous pattern was most frequent in the tumor center. Various distributions of E-cadherin expression depending on cancer progression were also found; E-cadherin expression in node-positive patients was lower in the tumor center and in the tumor invasive front, whereas, in patients with distant metastases, the expression of E-Cadherin was lower in the budding sites. In patients with higher TNM stages, E-cadherin expression was lower within the tumor (in the budding sites, tumor center, and invasive front). In tumors with lymphoid follicles, E-cadherin expression was higher in all localizations within the primary tumor. E-cadherin expression in the tumor center was also lower in tumors with some higher tumor budding parameters (areas of poorly differentiated components and poorly differentiated clusters). E-cadherin expression was found to be lower at the tumor center in younger individuals, at the budding sites in men, and at the surrounding lymph nodes in rectal tumors. Low E-cadherin expression appears to be a reliable indicator of higher cancer staging and progression. When assessing the advancement of cancer, apart from the TNM classification, it is beneficial to also consider the expression of E-cadherin. High tumor budding, the poverty of lymphoid follicles, and low E-cadherin expression analyzed simultaneously may contribute to a reliable assessment of colorectal cancer staging. These three histopathological features complement each other, and their investigation, together with conventional tumor staging and grading, may be very helpful in predicting the prognosis of colorectal cancer patients and qualifying them for the best treatment. The role of E-cadherin in the diagnosis and treatment of colorectal cancer, as a part of a personalized medicine strategy, still requires comprehensive, prospective clinical evaluations to precisely target the optimal therapies for the right patients at the right time.

Personal immune profiles: Diversity and prognostic value for oral tongue squamous cell carcinoma evaluated by comprehensive immune parameter analyses with multiplex immunofluorescence

ObjectivesUnderstanding the tumor immune microenvironment is becoming increasingly necessary for risk prediction and treatment selection. In particular, oral cancer has various immunosuppressive characteristics in the tumor microenvironment. Therefore, we comprehensively assessed the immune profiles of oral tongue squamous cell carcinoma (OTSCC). Materials and methodsMultiplex immunofluorescence and tissue imaging analyses were performed to evaluate immune profiles at the invasive tumor front of 60 OTSCC surgical specimens. We analyzed 58 immune parameters including the density and proportion (%) of total leukocytes (Leu) and T cells, six subsets of T and myeloid cells, and the expression of programmed cell death-1 (PD-1) and PD-1 ligand 1 (PD-L1). ResultsThe density, proportion, and location of CD45+ Leu, three T cell subsets (CD8+, Foxp3–CD4+ conventional, and Foxp3+CD4+ regulatory T cells), CD163-CD68+ M1 and CD163+CD68+ M2 macrophages, and neutrophils were highly variable at the individual level. The density and proportion of M2 macrophages were significantly lower in the T1 stage group. Risk prediction analyses for recurrence and/or metastasis (R/M) showed that R/M (+) T1 cases had significantly higher M2 density and percentages. ConclusionsThe immune profiles of OTSCC patients are diverse and cannot be predicted from clinicopathological information alone. The M2 macrophage abundance is a potential candidate biomarker for R/M in the early stage of OTSCC. Personal immune profiling may provide beneficial information for risk prediction and treatment selection.

Impact of vitamin d on the colon tumor immune microenvironment: Results of a randomized clinical trial of preoperative vitamin D supplementation in patients with stage I-III colon cancer.

3607 Background: Although the anti-tumor properties of vitamin D in colorectal cancer (CRC) have been broadly reported, its potential role as a tumor microenvironment (TME) immunomodulator has not been well established. Vitamin D receptor (VDR) and CYP27B1 (1-α-hydroxylase; required to convert vitamin D to its active form) are critical factors in the vitamin D signaling pathway and harbor prognostic significance for CRC. Pre-clinical studies suggest that higher stromal VDR expression enhances chemotherapy efficacy. To characterize the effects of vitamin D on the TME in humans, we conducted a randomized placebo-controlled trial of preoperative high-dose vitamin D supplementation in stage I-III colon cancer patients. Methods: Forty-two patients were randomized to receive vitamin D3 50,000 IU/day x 7 days followed by 10,000 IU/day versus placebo prior to resection. Paired pre-treatment biopsy specimens and post-treatment surgical resection specimens from 19 patients who completed the assigned treatment and had sufficient tissue for analysis were collected for spatially-resolved immune cell profiling and evaluation of VDR and CYP27B1 expression using custom multiplex immunofluorescence. Blood samples were collected pre- and post-treatment to assess plasma 25-hydroxyvitamin D [25(OH)D] levels. Linear mixed effects models were used to compare tissue parameters; plasma 25(OH)D level changes were assessed using the Wilcoxon rank-sum test. Results: Patients randomized to high-dose vitamin D had increased post-treatment plasma 25(OH)D (median 18.0 to 63.6 (ng/ml)) whereas placebo-randomized patients did not (median 19.1 to 18.6 (ng/ml)) ( p &lt;0.001). Analysis of both biopsy and resection tissue revealed highly heterogeneous VDR and CYP27B1 expression. VDR expression was highest in tumor cells, followed by immune and then stromal cells. Although resection tissue from vitamin D-treated patients showed lower VDR expression than specimens from placebo-treated patients in all cells ( p= 0.03), VDR was preferentially expressed by tumor cells at the tumor invasive front compared to tumor center in the vitamin D arm, while the opposite was observed in the placebo arm. Vitamin D supplementation did not alter CYP27B1 expression. While treatment did not change total immune cell density, resection tissue from vitamin D-treated patients had higher overall CD3+CD8+ T cell density ( p= 0.04), particularly CD3+CD8+ memory T cell density ( p= 0.02). Additionally, vitamin D supplementation led to greater co-localization between CD3+CD8+ T cells and tumor cells ( p&lt; 0.001) compared to placebo. Conclusions: In patients with stage I-III CRC, vitamin D supplementation led to changes in vitamin D pathway signaling and evidence of an anti-tumor effect in the TME. Further investigation into the underlying mechanism of action is warranted. Clinical trial information: NCT02172651 .

Spatial analysis of stromal signatures identifies invasive front carcinoma-associated fibroblasts as suppressors of anti-tumor immune response in esophageal cancer

BackgroundIncreasing evidence indicates that the tumor microenvironment (TME) is a crucial determinant of cancer progression. However, the clinical and pathobiological significance of stromal signatures in the TME, as a complex dynamic entity, is still unclear in esophageal squamous cell carcinoma (ESCC).MethodsHerein, we used single-cell transcriptome sequencing data, imaging mass cytometry (IMC) and multiplex immunofluorescence staining to characterize the stromal signatures in ESCC and evaluate their prognostic values in this aggressive disease. An automated quantitative pathology imaging system determined the locations of the lamina propria, stroma, and invasive front. Subsequently, IMC spatial analyses further uncovered spatial interaction and distribution. Additionally, bioinformatics analysis was performed to explore the TME remodeling mechanism in ESCC. To define a new molecular prognostic model, we calculated the risk score of each patient based on their TME signatures and pTNM stages.ResultsWe demonstrate that the presence of fibroblasts at the tumor invasive front was associated with the invasive depth and poor prognosis. Furthermore, the amount of α-smooth muscle actin (α-SMA)+ fibroblasts at the tumor invasive front positively correlated with the number of macrophages (MØs), but negatively correlated with that of tumor-infiltrating granzyme B+ immune cells, and CD4+ and CD8+ T cells. Spatial analyses uncovered a significant spatial interaction between α-SMA+ fibroblasts and CD163+ MØs in the TME, which resulted in spatially exclusive interactions to anti-tumor immune cells. We further validated the laminin and collagen signaling network contributions to TME remodeling. Moreover, compared with pTNM staging, a molecular prognostic model, based on expression of α-SMA+ fibroblasts at the invasive front, and CD163+ MØs, showed higher accuracy in predicting survival or recurrence in ESCC patients. Regression analysis confirmed this model is an independent predictor for survival, which also identifies a high-risk group of ESCC patients that can benefit from adjuvant therapy.ConclusionsOur newly defined biomarker signature may serve as a complement for current clinical risk stratification approaches and provide potential therapeutic targets for reversing the fibroblast-mediated immunosuppressive microenvironment.

Immune cell infiltration, tumour budding, and the p53 expression pattern are important predictors in penile squamous cell carcinoma: a retrospective study of 152 cases

Penile squamous cell carcinoma (pSCC) is a rare malignancy with a slowly increasing incidence and variable prognosis. Regional lymph node involvement signifies poor prognosis but represents a late sign, and more prognostic markers for effective patient risk stratification are urgently needed. In this retrospective study, 152 tumour samples with formalin-fixed, paraffin-embedded tissue were analysed for traditional pathological variables, tumour budding, p53, p16, and mismatch repair proteins (MMR) immunohistochemistry. The density of tumour lymphocytic infiltrate was also determined, using subjective evaluation by two pathologists (brisk/non-brisk/absent) and also using the immunoscore method, which categorised the cohort into five immunoscore groups according to the number of CD3+ and CD8+ T-cells in both the tumour centre and tumour invasion front. Only one case (0.6%) was MMR-deficient. Tumour budding count ≥5 tumour buds/20× power field and non-brisk/absent lymphocytic infiltrate were significant negative predictors of both the overall survival (OS) and cancer-specific survival (CSS), whereas a low immunoscore was a significant marker of shorter OS but not CSS. Advanced pT stage (3+4) was a significant marker of shorter CSS but not OS. In the multivariate analysis, high-grade budding was a significant parameter if adjusted for the patient's age and associated variables, except for the pN stage. The lymphocytic infiltrate retained its prognostic significance if adjusted for age and associated variables. The negative prognostic significance of the previously described parameters (lymphatic, venous, and perineural invasion, regional lymph node metastasis, and p53 mutated profile) were confirmed in our study. Grade, histological subtype, and HPV status (as determined by p16 immunohistochemistry) showed, surprisingly, little or no prognostic significance.

Cancer-associated fibroblasts in the invasive tumour front promote the metastasis of oral squamous cell carcinoma through MFAP5 upregulation

Cancer-associated fibroblasts (CAFs) are widely involved in the development and progression of tumours. As a direct junction between tumour and normal host tissue, the invasive tumour front can remodel host tissue to generate a microenvironment more suitable for tumour invasion. However, whether CAFs derived from the invasive front (CAFs-F) have a greater ability to promote tumour invasion than CAFs derived from the superficial tumour (CAFs-S) is unclear. In this study, we characterized primary CAFs from different spatial locations of tumours. We demonstrated that CAFs-F had an increased ability to promote oral squamous cell carcinoma (OSCC) proliferation and invasion in vitro and significantly enhanced tumour growth in vivo compared to CAFs-S. Mechanistically, transcriptome profiling analysis revealed that the expression of MFAP5, encoding microfibril associated protein 5, was dramatically increased in CAFs-F compared to CAFs-S, which further confirmed that the MFAP5 protein level was elevated in head and neck squamous cell carcinoma (HNSCC) and that this increase was correlated with poor survival. Genetic ablation of MFAP5 impaired the preinvasive capabilities of CAFs-F. Together, our findings demonstrated that CAFs-F had a greater ability to promote tumour invasion than CAFs-S and that MFAP5 might be involved in this process.