Periostin in lymph node pre-metastatic niches governs lymphatic endothelial cell functions and metastatic colonization.

Although lymph node (LN) metastasis is an important prognostic parameter in cervical cancer, the tissue remodeling at a pre-metastatic state is poorly documented in LNs. We here identified periostin (POSTN) as a component of non-metastatic LNs by applying proteomic analyses and computerized image quantifications on LNs of patients with cervical cancer. We provide evidence for remarkable modifications of POSTN and lymphatic vessel distributions and densities in non-metastatic sentinel and metastatic human LNs, when compared to distant non-metastatic LNs. POSTN deposition at a pre-metastatic stage was demonstrated in a pre-clinical murine model (the ear sponge assay). Its expression by fibroblastic LN cells was assessed by in situ hybridization and in vitro cultures. In vitro, POSTN promoted lymphatic endothelial cell functions and tumor cell proliferation. Accordingly, the in vivo injection of recombinant POSTN together with VEGF-C boosted the lymphangiogenic response, while the metastatic potential of tumor cells was drastically reduced using a POSTN blocking antibody. This translational study also supports the existence of an unprecedented dialog "in cascade", between the primary tumor and the first pelvic nodal relay in early cervical cancer, and subsequently from pelvic LN to para-aortic LNs in locally advanced cervical cancers. Collectively, this work highlights the association of POSTN deposition with lymphangiogenesis in LNs, and provides evidence for a key contribution of POSTN in promoting VEGF-C driven lymphangiogenesis and the seeding of metastatic cells.

Tumor resistance to ferroptosis driven by Stearoyl-CoA Desaturase-1 (SCD1) in cancer cells and Fatty Acid Biding Protein-4 (FABP4) in tumor microenvironment promote tumor recurrence.

PROBLEM: Tumor recurrence is a major clinical issue that represents the principal cause of cancer-related deaths, with few targetable common pathways. Mechanisms by which residual tumors persist and progress under a continuous shift between hypoxia-reoxygenation after neoadjuvent-therapy are unknown. In this study, we investigated the role of lipid metabolism and tumor redox balance in tumor recurrence. METHODS: Lipidomics, proteomics and mass spectrometry imaging approaches where applied to mouse tumor models of recurrence. Genetic and pharmacological inhibitions of lipid mediators in tumors were used in vivo and in functional assays in vitro. RESULTS: We found that stearoyl-CoA desaturase-1 (SCD1) expressed by cancer cells and fatty acid binding protein-4 (FABP4) produced by tumor endothelial cells (TECs) and adipocytes in the tumor microenvironment (TME) are essential for tumor relapse in response to tyrosine kinase inhibitors (TKI) and chemotherapy. SCD1 and FABP4 were also found upregulated in recurrent human breast cancer samples and correlated with worse prognosis of cancer patients with different types of tumors. Mechanistically, SCD1 leads to fatty acid (FA) desaturation and FABP4 derived from TEM enhances lipid droplet (LD) in cancer cells, which cooperatively protect from oxidative stress-induced ferroptosis. We revealed that lipid mobilization and desaturation elicit tumor intrinsic antioxidant and anti-ferroptotic resources for survival and regrowth in a harsh TME. Inhibition of lipid transport from TME by FABP4 inhibitor reduced tumor regrowth and by genetic - or by pharmacological - targeting SCD1 in vivo, tumor regrowth was abolished completely. CONCLUSION: This finding unveils that it is worth taking advantage of tumor lipid addiction, as a tumor vulnerability to design novel treatment strategy to prevent cancer recurrence.

EMT-Associated Heterogeneity in Circulating Tumor Cells: Sticky Friends on the Road to Metastasis.

Epithelial-mesenchymal transitions (EMTs) generate hybrid phenotypes with an enhanced ability to adapt to diverse microenvironments encountered during the metastatic spread. Accordingly, EMTs play a crucial role in the biology of circulating tumor cells (CTCs) and contribute to their heterogeneity. Here, we review major EMT-driven properties that may help hybrid Epithelial/Mesenchymal CTCs to survive in the bloodstream and accomplish early phases of metastatic colonization. We then discuss how interrogating EMT in CTCs as a companion biomarker could help refine cancer patient management, further supporting the relevance of CTCs in personalized medicine.

Tyrosine Kinase Inhibitors in Cancer: Breakthrough and Challenges of Targeted Therapy.

Receptor tyrosine kinases (RTKs) are key regulatory signaling proteins governing cancer cell growth and metastasis. During the last two decades, several molecules targeting RTKs were used in oncology as a first or second line therapy in different types of cancer. However, their effectiveness is limited by the appearance of resistance or adverse effects. In this review, we summarize the main features of RTKs and their inhibitors (RTKIs), their current use in oncology, and mechanisms of resistance. We also describe the technological advances of artificial intelligence, chemoproteomics, and microfluidics in elaborating powerful strategies that could be used in providing more efficient and selective small molecules inhibitors of RTKs. Finally, we discuss the interest of therapeutic combination of different RTKIs or with other molecules for personalized treatments, and the challenge for effective combination with less toxic and off-target effects.

Microproteomic Profiling of High-Grade Squamous Intraepithelial Lesion of the Cervix: Insight into Biological Mechanisms of Dysplasia and New Potential Diagnostic Markers.

PURPOSE: High-grade squamous intraepithelial lesion (HSIL) is a known precursor for squamous cell carcinoma of uterine cervix. Although it is known that SILs are associated to infection by human papillomavirus, downstream biological mechanisms are still poorly described. In this study, we compared the microproteomic profile of HSIL to normal tissues: ectocervix (ectoC) and endocervix (endoC). EXPERIMENTAL DESIGN: Tissue regions of endoC, ectoC, and HSlL were collected by laser microdissection (3500 cells each) from five patients. Samples were processed and analyzed using our recently developed laser microdissection-based microproteomic method. Tissues were compared in order to retrieve HSIL's proteomic profile. Potentially interesting proteins for pathology were stained by immunohistochemistry. RESULTS: We identified 3072 proteins among the fifteen samples and 2386 were quantified in at least four out of the five biological replicates of at least one tissue type. We found 236 proteins more abundant in HSIL. Gene ontology enrichments revealed mechanisms of DNA replication and RNA splicing. Despite the squamous nature of HSIL, a common signature between HSIL and endoC could be found. Finally, potential new markers could support diagnosis of dysplasia in SILs. CONCLUSION AND CLINICAL RELEVANCE: This microproteomic investigation of HSIL gives insights into the biology of cervical precancerous lesions.

High infiltration of CD68+ macrophages is associated with poor prognoses of head and neck squamous cell carcinoma patients and is influenced by human papillomavirus.

Incidence of human papillomavirus (HPV)-related head and neck squamous cell carcinomas (HNSCCs) has increased over the last few decades. The reaction of the host immune system to these tumors remains biologically complex. Here, we investigated CD68+ macrophage numbers, reporting the prognostic value in comparison to other risk factors. We also examined CD68+ macrophage infiltration during disease progression regarding the impact of HPV infection, and we studied the role of HPV16-E6/E7 oncoproteins in CD68+ macrophage recruitment. CD68+ macrophage numbers were evaluated in 10 cases of tumor-free peri-tumoral epithelia, 43 cases of low-grade dysplasia, 45 cases of high-grade dysplasia and 110 cases of carcinoma. Our in vivo model was developed in 80 C3H/HeN mice orthotopically injected with HPV16-E6, -E7 or -E6/E7-transfected SCC-VII cell lines. High CD68+ macrophage numbers in the intra-tumoral compartment were associated with shorter patient survival (recurrence-free survival: p = 0.001; overall survival: p = 0.01). Multivariate analyses reported that CD68+ macrophage infiltration and tumor stage were strong and independent prognostic factors of HNSCC. CD68+ macrophage numbers increased during HNSCC progression both in intra-epithelial (p < 0.001) and stromal compartments (p < 0.001). A higher density of CD68+ macrophages was observed in advanced stages (p = 0.004). Patients with transcriptionally active HPV infections had higher CD68+ macrophage density than did HPV-negative patients (p = 0.003). CD68+ macrophage infiltration was higher in HPV-E7+ and -E6/E7+ mouse tumors than in -E6+ tumors (p = 0.029 and p < 0.001). In conclusion, the extent of CD68+ macrophage infiltration is a significant prognostic factor for HNSCC patients. The recruitment of macrophages increases during disease progression and is influenced by the HPV virus.

MALDI Imaging-Guided Microproteomic Analyses of Heterogeneous Breast Tumors-A Pilot Study.

Matrix-assisted laser desorption/ionization (MALDI) imaging is an ideal tool to study intratumor heterogeneity (ITH) and its implication in prognostic stratification of patients. However, there are some drawbacks concerning protein identification. On the other hand, laser microdissection (LMD)-based microproteomics allows retrieving thousands of protein identifications from small tissue pieces. As a proof of concept, the authors combine these two complementary approaches to analyze heterogeneous regions in breast tumors. Invasive ductal breast cancer FFPE tissue sections from five patients are analyzed by MALDI imaging and the dataset is processed by segmentation. Heterogeneous regions within tumors are processed by LMD-based microproteomics, in duplicates. Liquid chromatography-tandem mass spectrometry data are classified by hierarchical clustering. Heterogeneous tissue regions are discriminated on the basis of their actual molecular heterogeneity. The dataset is correlated with MALDI imaging to identify m/z values discriminating heterogeneous regions. The molecular characterization of cell clones in tumors related to bad patient outcome could have great impact for pathology. A combined application of LMD-based microproteomics and MALDI imaging for ITH studies is presented.

A specific immune and lymphatic profile characterizes the pre-metastatic state of the sentinel lymph node in patients with early cervical cancer.

The lymph node (LN) pre-metastatic niche is faintly characterized in lymphophilic human neoplasia, although LN metastasis is considered as the strongest prognostic marker of patient survival. Due to its specific dissemination through a complex bilateral pelvic lymphatic system, early cervical cancer is a relevant candidate for investigating the early nodal metastatic process. In the present study, we analyzed in-depth both the lymphatic vasculature and the immune climate of pre-metastatic sentinel LN (SLN), in 48 cases of FIGO stage IB1 cervical neoplasms. An original digital image analysis methodology was used to objectively determine whole slide densities and spatial distributions of immunostained structures. We observed a marked increase in lymphatic vessel density (LVD) and a specific capsular and subcapsular distribution in pre-metastatic SLN when compared with non-sentinel counterparts. Such features persisted in the presence of nodal metastatic colonization. The inflammatory profile attested by CD8+, Foxp3, CD20 and PD-1expression was also significantly increased in pre-metastatic SLN. Remarkably, the densities of CD20+ B cells and PD-1 expressing germinal centers were positively correlated with LVD. All together, these data strongly support the existence of a pre-metastatic dialog between the primary tumor and the first nodal relay. Both lymphatic and immune responses contribute to the elaboration of a specific pre-metastatic microenvironment in human SLN. Moreover, this work provides evidence that, in the context of early cervical cancer, a pre-metastatic lymphangiogenesis occurs within the SLN (pre-metastatic niche) and is associated with a specific humoral immune response.

High stromal Foxp3-positive T cell number combined to tumor stage improved prognosis in head and neck squamous cell carcinoma.

OBJECTIVES: Head and neck squamous cell carcinomas (HNSCC), one of the most frequent cancers in the world, are largely infiltrated by inflammatory immune cells. Our aim was to evaluate the number of Foxp3+ T cells in HNSCC, reporting its prognostic power in comparison to other risk factors. MATERIAL AND METHODS: Our clinical series was composed of 21 tumor-free peri-tumoral epithelia, 49 low grade dysplasia, 43 high grade dysplasia and 110 carcinoma samples including some cases with HPV infection. In vivo experiments were conducted on 80 C3H/HeN mice which were orthotopically injected with SCCVII CT, E7, E6 and E6/E7 cell lines. RESULTS: Foxp3+ T cell infiltration increased with tumor progression from normal epithelia, dysplasia to carcinoma and the increase is more important in HPV+ patients than in negative ones. Animal experiments revealed that E7 oncoprotein expression was significantly associated with an increase in Foxp3+ T cell recruitment in tumor, a delay in tumor onset and improved animal survival. Univariate Cox regression analyses demonstrated that high Foxp3+ T cell number in stromal compartment is associated with longer patient recurrence-free and overall survivals. Foxp3+ T cell number improved the prognostic value of tumor stage. Multivariate analyses reported that stromal Foxp3+ T cell number is a strong prognostic factor independent of classical risk factors such as tobacco, alcohol, and HPV status. CONCLUSION: Foxp3+ T cell number is a significant prognostic factor for HNSCC, improving the tumor stage, and that viral E7 may play a role in the Foxp3+ T cell infiltration to the tumor.

Langerhans cell number is a strong and independent prognostic factor for head and neck squamous cell carcinomas.

OBJECTIVES: Head and neck squamous cell carcinomas (HNSCCs) exhibit great biological heterogeneity and relatively poor prognosis. Tobacco and alcohol consumption is involved in the cause of the majority of these cancers, but over the last several years, Human Papilloma Virus (HPV) infection has increased specifically in oropharyngeal cancers and become an additional risk factor. Here, we evaluated the number of Langerhans cells (LCs) in HNSCC and reporting its prognostic power in comparison to other risk factors. MATERIALS AND METHODS: Our clinical series was composed of 25 tumor-free peritumoral epithelium, 64 low-grade dysplasia, 54 high-grade dysplasia and 125 carcinoma samples. HPV was detected by E6/E7 qPCR and p16 immunohistochemistry. CD1a-positive LCs were counted in intra-tumoral and stromal compartments as well as lymph nodes. MIP-3α was assessed in carcinomas using immunohistochemistry. RESULTS: Univariate Cox regression analyses demonstrated that high LC number is associated with longer recurrence-free survival in both intra-tumoral and stromal compartments and longer overall survival in stromal compartment. Tobacco and alcohol habits, but not HPV status, are also correlated with poor prognoses in terms of recurrence. Multivariate analyses reported stromal LC number as a strong prognostic factor independent of tobacco, alcohol and HPV status. Moreover, LC number is higher in tumors and invaded lymph nodes than dysplastic lesions but it decreases in HPV-positive cancer patients. Further, LC number correlates with MIP-3α expression. CONCLUSION: These findings suggest that LC number is a significant and independent prognostic factor for HNSCC. LC infiltration is increased in cancer lesions but decrease with HPV infection.

MALDI mass spectrometry imaging: A cutting-edge tool for fundamental and clinical histopathology.

Histopathological diagnoses have been done in the last century based on hematoxylin and eosin staining. These methods were complemented by histochemistry, electron microscopy, immunohistochemistry (IHC), and molecular techniques. Mass spectrometry (MS) methods allow the thorough examination of various biocompounds in extracts and tissue sections. Today, mass spectrometry imaging (MSI), and especially matrix-assisted laser desorption ionization (MALDI) imaging links classical histology and molecular analyses. Direct mapping is a major advantage of the combination of molecular profiling and imaging. MSI can be considered as a cutting edge approach for molecular detection of proteins, peptides, carbohydrates, lipids, and small molecules in tissues. This review covers the detection of various biomolecules in histopathological sections by MSI. Proteomic methods will be introduced into clinical histopathology within the next few years.

A laser microdissection-based workflow for FFPE tissue microproteomics: Important considerations for small sample processing.

Proteomic methods are today widely applied to formalin-fixed paraffin-embedded (FFPE) tissue samples for several applications in research, especially in molecular pathology. To date, there is an unmet need for the analysis of small tissue samples, such as for early cancerous lesions. Indeed, no method has yet been proposed for the reproducible processing of small FFPE tissue samples to allow biomarker discovery. In this work, we tested several procedures to process laser microdissected tissue pieces bearing less than 3000 cells. Combined with appropriate settings for liquid chromatography mass spectrometry-mass spectrometry (LC-MS/MS) analysis, a citric acid antigen retrieval (CAAR)-based procedure was established, allowing to identify more than 1400 proteins from a single microdissected breast cancer tissue biopsy. This work demonstrates important considerations concerning the handling and processing of laser microdissected tissue samples of extremely limited size, in the process opening new perspectives in molecular pathology. A proof of the proposed method for biomarker discovery, with respect to these specific handling considerations, is illustrated using the differential proteomic analysis of invasive breast carcinoma of no special type and invasive lobular triple-negative breast cancer tissues. This work will be of utmost importance for early biomarker discovery or in support of matrix-assisted laser desorption/ionization (MALDI) imaging for microproteomics from small regions of interest.

The importance of the tumor microenvironment in the therapeutic management of cancer.

Tumor prognosis is generally defined by various tumor parameters. However, it is well known that paracrine, endocrine and cell-cell interactions between the tumor and its microenvironment contribute to its growth. The tumor microenvironment (TME) can also influence disease prognosis and is likely to be considered as an important prognostic factor. In addition, conventional therapies can influence the microenvironment and antitumor immunity. Similarly, the TME will influence the effectiveness of therapy. The purpose of this review is to demonstrate how TME is important in therapeutic management. Key interactions between TME and different cancer therapies as well as their current clinical consequences have been described. More research is needed to establish the important network between tumor cells and their environment to highlight their relationships with conventional therapies and develop global therapeutic strategies.

Tissue proteomics for the next decade? Towards a molecular dimension in histology.

The concept of tissues appeared more than 200 years ago, since textures and attendant differences were described within the whole organism components. Instrumental developments in optics and biochemistry subsequently paved the way to transition from classical to molecular histology in order to decipher the molecular contexts associated with physiological or pathological development or function of a tissue. In 1941, Coons and colleagues performed the first systematic integrated examination of classical histology and biochemistry when his team localized pneumonia antigens in infected tissue sections. Most recently, in the early 21(st) century, mass spectrometry (MS) has progressively become one of the most valuable tools to analyze biomolecular compounds. Currently, sampling methods, biochemical procedures, and MS instrumentations allow scientists to perform "in depth" analysis of the protein content of any type of tissue of interest. This article reviews the salient issues in proteomics analysis of tissues. We first outline technical and analytical considerations for sampling and biochemical processing of tissues and subsequently the instrumental possibilities for proteomics analysis such as shotgun proteomics in an anatomical context. Specific attention concerns formalin fixed and paraffin embedded (FFPE) tissues that are potential "gold mines" for histopathological investigations. In all, the matrix assisted laser desorption/ionization (MALDI) MS imaging, which allows for differential mapping of hundreds of compounds on a tissue section, is currently the most striking evidence of linkage and transition between "classical" and "molecular" histology. Tissue proteomics represents a veritable field of research and investment activity for modern biomarker discovery and development for the next decade.