Regulation of Rubisco activity by interaction with chloroplast metabolites.

Rubisco activity is highly regulated and frequently limits carbon assimilation in crop plants. In the chloroplast, various metabolites can inhibit or modulate Rubisco activity by binding to its catalytic or allosteric sites, but this regulation is complex and still poorly understood. Using rice Rubisco, we characterised the impact of various chloroplast metabolites which could interact with Rubisco and modulate its activity, including photorespiratory intermediates, carbohydrates, amino acids; as well as specific sugar-phosphates known to inhibit Rubisco activity - CABP (2-carboxy-D-arabinitol 1,5-bisphosphate) and CA1P (2-carboxy-D-arabinitol 1-phosphate) through in vitro enzymatic assays and molecular docking analysis. Most metabolites did not directly affect Rubisco in vitro activity under both saturating and limiting concentrations of Rubisco substrates, CO2 and RuBP (ribulose-1,5-bisphosphate). As expected, Rubisco activity was strongly inhibited in the presence of CABP and CA1P. High physiologically relevant concentrations of the carboxylation product 3-PGA (3-phosphoglyceric acid) decreased Rubisco activity by up to 30%. High concentrations of the photosynthetically derived hexose phosphates fructose 6-phosphate (F6P) and glucose 6-phosphate (G6P) slightly reduced Rubisco activity under limiting CO2 and RuBP concentrations. Biochemical measurements of the apparent Vmax and Km for CO2 and RuBP (at atmospheric O2 concentration) and docking interactions analysis suggest that CABP/CA1P and 3-PGA inhibit Rubisco activity by binding tightly and loosely, respectively, to its catalytic sites (i.e., competing with the substrate RuBP). These findings will aid the design and biochemical modelling of new strategies to improve the regulation of Rubisco activity and enhance the efficiency and sustainability of carbon assimilation in rice.

Epigenetic regulation of the tumor microenvironment: A leading force driving pancreatic cancer.

Dysregulation of the epigenomic landscape of tumor cells has been implicated in the pathogenesis of pancreatic cancer. However, these alterations are not only restricted to neoplastic cells. The behavior of other cell populations in the tumor stroma such as cancer-associated fibroblasts, immune cells, and others are mostly regulated by epigenetic pathways. Here, we present an overview of the main cellular and acellular components of the pancreatic cancer tumor microenvironment and discuss how the epigenetic mechanisms operate at different levels in the stroma to establish a differential gene expression to regulate distinct cellular phenotypes contributing to pancreatic tumorigenesis.

Fabrication of GelMA - Agarose Based 3D Bioprinted Photocurable Hydrogel with In Vitro Cytocompatibility and Cells Mirroring Natural Keratocytes for Corneal Stromal Regeneration.

The complex anatomy of the cornea and the subsequent keratocyte-fibroblast transition have always made corneal stromal regeneration difficult. Recently, 3D printing has received considerable attention in terms of fabrication of scaffolds with precise dimension and pattern. In the current work, 3D printable polymer hydrogels made of GelMA/agarose are formulated and its rheological properties are evaluated. Despite the variation in agarose content, both the hydrogels exhibited G'>G'' modulus. A prototype for 3D stromal model is created using Solid Works software, mimicking the anatomy of an adult cornea. The fabrication of 3D-printed hydrogels is performed using pneumatic extrusion. The FTIR analysis speculated that the hydrogel is well crosslinked and established strong hydrogen bonding with each other, thus contributing to improved thermal and structural stability. The MTT analysis revealed a higher rate of cell proliferation on the hydrogels. The optical analysis carried out on the 14th day of incubation revealed that the hydrogels exhibit transparency matching with natural corneal stromal tissue. Specific protein marker expression confirmed the keratocyte phenotype and showed that the cells do not undergo terminal differentiation into stromal fibroblasts. The findings of this work point to the potential of GelMA/A hydrogels as a novel biomaterial for corneal stromal tissue engineering.

A Microdissection Protocol for Proteogenomic Analysis of Histological Sections to Advance Drug Development.

Combining proteogenomics with laser capture microdissection (LCM) in cancer research offers a targeted way to explore the intricate interactions between tumor cells and the different microenvironment components. This is especially important for immuno-oncology (IO) research where improvements in the predictability of IO-based drugs are sorely needed, and depends on a better understanding of the spatial relationships involving the tumor, blood supply, and immune cell interactions, in the context of their associated microenvironments. LCM is used to isolate and obtain distinct histological cell types, which may be routinely performed on complex and heterogeneous solid tumor specimens. Once cells have been captured, nucleic acids and proteins may be extracted for in-depth multimodality molecular profiling assays. Optimizing the minute tissue quantities from LCM captured cells is challenging. Following the isolation of nucleic acids, RNA-seq may be performed for gene expression and DNA sequencing performed for the discovery and analysis of actionable mutations, copy number variation, methylation profiles, etc. However, there remains a need for highly sensitive proteomic methods targeting small-sized samples. A significant part of this protocol is an enhanced liquid chromatography mass spectrometry (LC-MS) analysis of micro-scale and/or nano-scale tissue sections. This is achieved with a silver-stained one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D-SDS-PAGE) approach developed for LC-MS analysis of fresh-frozen tissue specimens obtained via LCM. Included is a detailed in-gel digestion method adjusted and specifically designed to maximize the proteome coverage from amount-limited LCM samples to better facilitate in-depth molecular profiling. Described is a proteogenomic approach leveraged from microdissected fresh frozen tissue. The protocols may also be applicable to other types of specimens having limited nucleic acids, protein quantity, and/or sample volume.

The tumor-stroma ratio in giant cell tumor of bone: associations with the immune microenvironment and responsiveness to denosumab treatment.

BACKGROUND: Currently, there is limited understanding regarding the clinical significance of the tumor-stroma ratio (TSR) in giant cell tumor of bone (GCTB). Hence, we aimed to investigate the distribution of TSR in GCTB and explore its correlation with various clinicopathologic factors, immune microenvironment, survival prognosis, and denosumab treatment responsiveness. METHODS: We conducted a multicenter cohort study comprising 426 GCTB patients treated at four centers. TSR was evaluated on hematoxylin and eosin-stained and immunofluorescent sections of tumor specimens. Immunohistochemistry was performed to assess CD3+, CD4+, CD8+, CD20+, PD-1+, PD-L1+, and FoxP3+ TIL subtypes as well as Ki-67 expression levels in 426 tissue specimens. These parameters were then analyzed for their correlations with patient outcomes [local recurrence-free survival (LRFS) and overall survival (OS)], clinicopathological features, and denosumab treatment responsiveness. RESULTS: Low TSR was significantly associated with poor LRFS and OS in both cohorts. Furthermore, TSR was also correlated with multiple clinicopathological features, TIL subtype expression, and denosumab treatment responsiveness. TSR demonstrated similar predictive capabilities as the conventional Campanacci staging system for predicting patients' LRFS and OS. CONCLUSION: The results of this study provide evidence supporting the use of TSR as a reliable prognostic tool in GCTB and as a predictor of denosumab treatment responsiveness. These findings may aid in developing individualized treatment strategies for GCTB patients in the future.

Insights into atypical segmental layer thicknesses and phase retardation in thick corneas using ultrahigh-resolution polarization-sensitive optical coherence tomography.

BACKGROUND: Accurately assessing corneal structural status is challenging when thickness deviates from the average. Polarization-sensitive optical coherence tomography (PS-OCT) measures tissue-specific polarization changes, providing additional contrast for accurate segmentations and aids in phase retardation (PR) measurements. Previous studies have shown PR's effectiveness in identifying sub-clinical keratoconus (KC) in asymmetric cases. Thus, this study aims to assess PR distribution in thick corneas with and without KC. METHODS: In this retrospective and cross-sectional study, 45 thick corneas from 30 Asian-Indian subjects, categorized into healthy (n = 26) and KC (n = 19) groups were analyzed. All eyes underwent standard clinical evaluations, tomographic assessments, and corneal biomechanics measurements. PR and individual layer thicknesses were measured using custom-designed ultrahigh-resolution PS-OCT. PR en-face maps were generated. Individual layer thicknesses and PR analysis was conducted across multiple zones, extending up to 8-10 mm in diameter. All eyes in the study had not undergone interventions, received topical medications, or had previous corneal disease history. RESULTS: Significant differences were found in spherical and cylindrical powers, keratometry, pachymetry, and biomechanical indices (all P < 0.01). Thickness profiles from PS-OCT showed significant differences in the 4-8 mm zones only. Bowman's layer thickness significantly differed only in the central 2 mm zone (P = 0.02). The median PR values showed marginal differences in the central 2 mm zone (P = 0.0565). Additionally, there were significant differences observed in the 2-4 mm and 4-6 mm zones (P = 0.0274 and P = 0.0456, respectively). KC eyes exhibited an atypical PR distribution and corneal thinning, while normal eyes maintained a uniform Bowman's layer thickness and PR maps with larger areas of higher PR. CONCLUSION: The study revealed distinctive PR distribution in thick corneas among healthy and KC groups. Using an ultrahigh-resolution PS-OCT the significance of Bowman's layer thickness in these groups was also emphasized. The study offered potential improvements in clinical diagnostics by enhancing our understanding of corneal structure and its altered function.

Bone Marrow Stroma Co-cultivation Model of Breast Cancer Dormancy.

Breast cancer cells metastasize to the bone marrow before a primary tumor is detected. Most micrometastases die in this hostile microenvironment, but some survive and enter a state of dormancy and chemoresistance due to their close interaction with cells in the bone marrow hematopoietic niche. Over many years, some of the cells reawaken and result in metastatic disease that cannot be cured. Analyzing the cellular and molecular interactions between cancer and bone marrow niche cells requires relevant models that can be manipulated and studied. Generation of bone marrow stroma cultures in vitro permits the formation of cellular monolayers upon which breast cancer cells can be co-cultivated and their behavior interrogated using a variety of techniques. This manuscript describes the basic techniques for generating bone marrow stromal monolayers, co-incubating cancer cells and determining the effects on cancer cell proliferation and molecular signaling.

A Rare Case of Pleural Epithelioid Mesothelioma With a Prominent Myxoid Stroma Reported With Morphology, Fluorescent In Situ Hybridization, and Ultrastructural Findings.

Herein, we report a rare case of pleural epithelioid malignant mesothelioma with a prominent myxoid stroma. To date, detailed morphological or molecular pathological findings have not been reported for this type of tumor. Hence, we aimed to describe the cytological, histological, immuno-cytohistological, electron-microscopic, and molecular pathological findings using fluorescence in situ hybridization (FISH) in such a case. The patient was a male in his mid-sixties with a history of asbestos exposure and had originally visited the hospital with a persistent cough and fever. Chest radiography revealed left pleural effusion, and laboratory examination revealed a high titer for hyaluronic acid in the effusion. Additionally, computed tomography revealed diffuse multinodular or cystic lesions in the left parietal pleura, and pleural effusion cytology revealed large epithelioid cells with mild nuclear atypia, which were considered reactive mesothelial cells. Cytologically, Giemsa staining revealed that these cells harbored variously sized intracytoplasmic vacuoles that were Alcian-blue-positive, suggesting hyaluronan production. Biopsy revealed large epithelioid cells that loosely proliferated against a prominent myxoid background. These cells were immuno-positive for calretinin, Wilms' tumor 1, D2-40, vimentin, and cytokeratin AE1/AE3 but not for carcinoembryonic antigen, Ber-EP4, or desmin. BRCA 1 associated protein 1 immunostaining showed nuclear loss, and FISH showed homozygous deletion of cyclin-dependent kinase inhibitor 2A (p16) on chromosome 9p21. Based on these findings, the lesion was diagnosed as an epithelioid mesothelioma with a prominent myxoid stroma. Electron-microscopy demonstrated a dense microvillus pattern on the surface of the tumor cells, indicating a mesothelial cell origin, and variously sized vacuoles in the cytoplasm, confirming the presence of intracytoplasmic vacuoles demonstrated on cytology. The tumor tissues obtained during surgery harbored prominent myxoid stroma, which proved that the present tumor was consistent with this type of mesothelioma. After informed consent was obtained, the patient and family wished for total resection of the tumor and postoperative chemotherapy, and the patient eventually died eight months after surgery.

Tissue Mechanics and Hedgehog Signaling Crosstalk as a Key Epithelial-Stromal Interplay in Cancer Development.

Epithelial-stromal interplay through chemomechanical cues from cells and matrix propels cancer progression. Elevated tissue stiffness in potentially malignant tissues suggests a link between matrix stiffness and enhanced tumor growth. In this study, employing chronic oral/esophageal injury and cancer models, it is demonstrated that epithelial-stromal interplay through matrix stiffness and Hedgehog (Hh) signaling is key in compounding cancer development. Epithelial cells actively interact with fibroblasts, exchanging mechanoresponsive signals during the precancerous stage. Specifically, epithelial cells release Sonic Hh, activating fibroblasts to produce matrix proteins and remodeling enzymes, resulting in tissue stiffening. Subsequently, basal epithelial cells adjacent to the stiffened tissue become proliferative and undergo epithelial-to-mesenchymal transition, acquiring migratory and invasive properties, thereby promoting invasive tumor growth. Notably, transcriptomic programs of oncogenic GLI2, mechano-activated by actin cytoskeletal tension, govern this process, elucidating the crucial role of non-canonical GLI2 activation in orchestrating the proliferation and mesenchymal transition of epithelial cells. Furthermore, pharmacological intervention targeting tissue stiffening proves highly effective in slowing cancer progression. These findings underscore the impact of epithelial-stromal interplay through chemo-mechanical (Hh-stiffness) signaling in cancer development, and suggest that targeting tissue stiffness holds promise as a strategy to disrupt chemo-mechanical feedback, enabling effective cancer treatment.

Functional and clinical roles of stromal PDGF receptors in tumor biology.

PDGF receptors play pivotal roles in both developmental and physiological processes through the regulation of mesenchymal cells involved in paracrine instructive interactions with epithelial or endothelial cells. Tumor biology studies, alongside analyses of patient tissue samples, provide strong indications that the PDGF signaling pathways are also critical in various types of human cancer. This review summarizes experimental findings and correlative studies, which have explored the biological mechanisms and clinical relevance of PDGFRs in mesenchymal cells of the tumor microenvironment. Collectively, these studies support the overall concept that the PDGF system is a critical regulator of tumor growth, metastasis, and drug efficacy, suggesting yet unexploited targeting opportunities. The inter-patient variability in stromal PDGFR expression, as being linked to prognosis and treatment responses, not only indicates the need for stratified approaches in upcoming therapeutic investigations but also implies the potential for the development of PDGFRs as biomarkers of clinical utility, interestingly also in settings outside PDGFR-directed treatments.

Enhancing Neoadjuvant Virotherapy's Effectiveness by Targeting Stroma to Improve Resectability in Pancreatic Cancer.

About one-fourth of patients with pancreatic ductal adenocarcinoma (PDAC) are categorized as borderline resectable (BR) or locally advanced (LA). Chemotherapy and radiation therapy have not yielded the anticipated outcomes in curing patients with BR/LA PDAC. The surgical resection of these tumors presents challenges owing to the unpredictability of the resection margin, involvement of vasculature with the tumor, the likelihood of occult metastasis, a higher ratio of positive lymph nodes, and the relatively larger size of tumor nodules. Oncolytic virotherapy has shown promising activity in preclinical PDAC models. Unfortunately, the desmoplastic stroma within the PDAC tumor microenvironment establishes a barrier, hindering the infiltration of oncolytic viruses and various therapeutic drugs-such as antibodies, adoptive cell therapy agents, and chemotherapeutic agents-in reaching the tumor site. Recently, a growing emphasis has been placed on targeting major acellular components of tumor stroma, such as hyaluronic acid and collagen, to enhance drug penetration. Oncolytic viruses can be engineered to express proteolytic enzymes that cleave hyaluronic acid and collagen into smaller polypeptides, thereby softening the desmoplastic stroma, ultimately leading to increased viral distribution along with increased oncolysis and subsequent tumor size regression. This approach may offer new possibilities to improve the resectability of patients diagnosed with BR and LA PDAC.

Targeting the pancreatic tumor microenvironment by plant-derived products and their nanoformulations.

Pancreatic cancer remains a significant health issue with limited treatment options. The tumor stroma, a complex environment made up of different cells and proteins, plays a crucial role in tumor growth and chemoresistance. Targeting tumor stroma, consisting of diverse non-tumor cells such as fibroblasts, extracellular matrix (ECM), immune cells, and also pre-vascular cells is encouraging for remodeling solid cancers, such as pancreatic cancer. Remodeling the stroma of pancreas tumors can be suggested as a strategy for reducing resistance to chemo/immunotherapy. Several studies have shown that phytochemicals from plants can affect the tumor environment and have anti-cancer properties. By targeting key pathways involved in stromal activation, phytochemicals may disrupt communication between the tumor and stroma and make tumor cells more sensitive to different treatments. Additionally, phytochemicals have immunomodulatory and anti-angiogenic properties, all of which contribute to their potential in treating pancreatic cancer. This review will provide a detailed look at how phytochemicals impact the tumor stroma and their effects on pancreatic tumor growth, spread, and response to treatment. It will also explore the potential of combining phytochemicals with other treatment options like chemotherapy, immunotherapy, and radiation.

Combination of DNA ploidy, stroma, and nucleotyping predicting prognosis and tailoring adjuvant chemotherapy duration in stage III colon cancer.

Introduction: DNA ploidy (P), stroma fraction (S), and nucleotyping (N) collectively known as PSN, have proven prognostic accuracy in stage II colorectal cancer (CRC). However, few studies have reported on the prognostic value of the PSN panel in stage III colon cancer patients receiving capecitabine and oxaliplatin adjuvant chemotherapy. Objectives: This study aimed to validate PSN's prognostic impact on stage III colon cancer, identifying candidates for optimized adjuvant chemotherapy duration. Design: A retrospective analysis was conducted on a cohort of stage III colon cancer patients from April 2008 to June 2020. Methods: Postoperative pathological samples from stage III colon cancer patients who underwent radical surgery and postoperative adjuvant chemotherapy at Sun Yat-sen University Cancer Center were retrospectively collected. Automated digital imaging assessed PSN, categorizing risk groups. Kaplan-Meier, Cox regression, and time-dependent receiver operating characteristic analysis compared model validity. Results: Significant differences in 5-year disease-free survival (DFS) and overall survival (OS) were noted among PSN-based low-, moderate-, and high-risk groups (DFS: 92.10% versus 83.62% versus 79.80%, p = 0.029; OS: 96.69% versus 93.99% versus 90.12%, p = 0.016). PSN emerged as an independent prognostic factor for DFS [hazard ratio (HR) = 1.409, 95% confidence interval (CI): 1.002-1.981, p = 0.049] and OS (HR = 1.720, 95% CI: 1.127-2.624, p = 0.012). The PSN model, incorporating perineural invasion and tumor location, displayed superior area under the curve for 5-year (0.692 versus 0.553, p = 0.020) and 10-year (0.694 versus 0.532, p = 0.006) DFS than TNM stage. In the PSN high-risk group, completing eight cycles of adjuvant chemotherapy significantly improved 5-year DFS and OS compared to four to seven cycles (DFS: 89.43% versus 71.52%, p = 0.026; OS: 96.77% versus 85.46%, p = 0.007). Conclusion: The PSN panel effectively stratifies stage III colon cancer, aiding in optimized adjuvant chemotherapy duration determination.

Dietary vitamin D intake and risk of colorectal cancer according to vitamin D receptor expression in tumors and their surrounding stroma.

BACKGROUND: Colorectal Cancer (CRC) has been molecularly classified into several subtypes according to tumor, stromal, and immune components. Here, we investigated whether the preventive effect of vitamin D on CRC varies with subtypes defined by Vitamin D receptor (VDR) expression in tumors and their surrounding stroma, along with the association of somatic mutations in CRC. METHODS: In a population-based prospective study of 22,743 Japanese participants, VDR expression levels in tumors and their surrounding stroma were defined in 507 cases of newly diagnosed CRC using immunohistochemistry. Hazard ratios of CRC and its subtypes according to dietary vitamin D intake were estimated using multivariable Cox proportional hazards models. RESULTS: Dietary vitamin D intake was not associated with CRC or its subtypes defined by VDR expression in tumors. However, an inverse association was observed for CRC with high VDR expression in the stroma (the highest tertile vs the lowest tertile: 0.46 [0.23-0.94], Ptrend = 0.03), but not for CRC with low VDR expression in the stroma (Pheterogeneity = 0.02). Furthermore, CRC with high VDR expression in the stroma had more somatic TP53 and BRAF mutations and fewer APC mutations than those with low VDR expression in the stroma. CONCLUSIONS: This study provides the first evidence that the preventive effect of vitamin D on CRC depends on VDR expression in the stroma rather than in the tumors. CRC with high VDR expression in the stroma is likely to develop through a part of the serrated polyp pathway, which tends to occur with BRAF but not with APC mutations.

Elimination of radiation-induced senescent cancer cells and stromal cells in vitro by near-infrared photoimmunotherapy.

INTRODUCTION: Therapy-induced senescent cancer and stromal cells secrete cytokines and growth factors to promote tumor progression. Therefore, senescent cells may be novel targets for tumor treatment. Near-infrared photoimmunotherapy (NIR-PIT) is a highly tumor-selective therapy that employs conjugates of a molecular-targeting antibody and photoabsorber. Thus, NIR-PIT has the potential to be applied as a novel senolytic therapy. This study aims to investigate the efficacy of NIR-PIT treatment on senescent cancer and stromal cells. METHODS: Two cancer cell lines (human lung adenocarcinoma A549 cells and human pancreatic cancer MIA PaCa-2 cells) and two normal cell lines (mouse fibroblast transfected with human epidermal growth factor receptor 2 [HER2] cells and human fibroblast WI38 cells) were used. The cytotoxicity of NIR-PIT was evaluated using anti-epidermal growth factor receptor (EGFR) antibody panitumumab and anti-HER2 antibody transtuzumab. RESULTS: Cellular senescence was induced in A549 and MIA PaCa-2 cells by 10 Gy γ-irradiation. The up-regulation of cellular senescence markers and characteristic morphological changes in senescent cells, including enlargement, flattening, and multinucleation, were observed in cancer cells after 5 days of γ-irradiation. Then, NIR-PIT targeting EGFR was performed on these senescent cancer cells. The NIR-PIT induced morphological changes, including bleb formation, swelling, and the inflow of extracellular fluid, and induced a significant decrease in cellular viability. These results suggested that NIR-PIT may induce cytotoxicity using the same mechanism in senescent cancer cells. In addition, similar morphological changes were also induced in radiation-induced senescent 3T3-HER2 fibroblasts by NIR-PIT targeting human epidermal growth factor receptor 2. CONCLUSION: NIR-PIT eliminates both senescent cancer and stromal cells in vitro suggesting it may be a novel strategy for tumor treatment.

Identifying the Morphological and Molecular Features of a Cell-Based Orthotopic Pancreatic Cancer Mouse Model during Growth over Time.

Pancreatic ductal adenocarcinoma (PDAC), characterized by hypovascularity, hypoxia, and desmoplastic stroma is one of the deadliest malignancies in humans, with a 5-year survival rate of only 7%. The anatomical location of the pancreas and lack of symptoms in patients with early onset of disease accounts for late diagnosis. Consequently, 85% of patients present with non-resectable, locally advanced, or advanced metastatic disease at diagnosis and rely on alternative therapies such as chemotherapy, immunotherapy, and others. The response to these therapies highly depends on the stage of disease at the start of therapy. It is, therefore, vital to consider the stages of PDAC models in preclinical studies when testing new therapeutics and treatment modalities. We report a standardized induction of cell-based orthotopic pancreatic cancer models in mice and the identification of vital features of their progression by ultrasound imaging and histological analysis of the level of pancreatic stellate cells, mature fibroblasts, and collagen. The results highlight that early-stage primary tumors are secluded in the pancreas and advance towards infiltrating the omentum at week 5-7 post implantation of the BxPC-3 and Panc-1 models investigated. Late stages show extensive growth, the infiltration of the omentum and/or stomach wall, metastases, augmented fibroblasts, and collagen levels. The findings can serve as suggestions for defining growth parameter-based stages of orthotopic pancreatic cancer models for the preclinical testing of drug efficacy in the future.

Prognostic value of tumor-stroma ratio in clear cell renal cell carcinoma after surgery.

Background: Clear cell renal cell carcinoma (ccRCC) is one of the most common urological tumors, and its incidence is increasing year by year. Tumor stroma ratio (TSR) can reflect the amount of stromal component around tumor cells, and can independently predict the prognosis of tumor. This study aims to evaluate the prognostic value of TSR in ccRCC patients. Methods: From January 2010 to December 2015, clinical and histopathological data of patients with ccRCC patients who underwent surgical operation were collected. Using TSR (50%) as the cut-off value, the patients were divided into low-TSR group (<50%) and high-TSR group (≥50%). The clinicopathological characteristics and survival status of patients were compared between the two groups. Univariate and multivariate analyses were used to identify the prognostic factors for overall survival (OS), cancer-specific survival (CSS), metastasis-free survival (MFS). Results: The mean age of 569 patients was 56.84±12.76 years old. There were 401 males and 168 females. According to the TSR, patients were divided into low-TSR group (n=333, 58.5%) and high-TSR group (n=236, 41.5%). The median follow-up time was 67.0 months (interquartile range, 33.0-72.0 months). The 5-year OS, CSS and MFS were 91.2%, 94.6% and 91.0%, respectively. The 5-year OS, CSS and MFS were 84.2%,89.7% and 82.7% in the high-TSR group and 96.1%, 98.0% and 96.0% in the low-TSR group (P<0.05). Multivariate analysis showed that age >60 years [hazard ratio (HR) =2.455, 95% confidence interval (CI): 1.292-4.668, P=0.006), tumor grade (HR =6.580, 95% CI: 3.276-13.216, P<0.001) and TSR (HR =2.611, 95% CI: 1.265-5.387, P=0.009) were independent prognostic factors for OS. Multivariate analysis showed that tumor stage (HR =3.213, 95% CI: 1.437-7.184, P=0.004), tumor grade (HR =6.102, 95% CI: 2.664-13.976, P<0.001) and TSR (HR =2.653, 95% CI: 1.063-6.621, P=0.03) were independent prognostic factors for CSS. Multivariate analysis showed that tumor stage (HR =4.805, 95% CI: 2.677-8.624, P<0.001), tumor grade (HR =6.423, 95% CI: 3.432-12.020, P<0.001), hemorrhage (HR =0.514, 95% CI: 0.265-0.996, P=0.049) and TSR (HR =2.370, 95% CI: 1.264-4.443, P=0.007) were independent prognostic factors for MFS. Conclusions: TSR is a new independent prognostic risk factor for ccRCC patients. The assessment of TSR is simple and cost-effective, and it is a useful supplement added to the pathological evaluation system.

Histological pattern of tumor inflammation and stromal density correlate with patient demographics and immuno-oncologic transcriptional profile in oral squamous cell carcinoma.

Introduction: Oral squamous cell carcinoma (OSCC) is the most prevalent oral malignancy, with emerging interest in the characterization of its tumor microenvironment. Herein, we present a comprehensive histological analysis of OSCC stromal density and inflammation and their relationship with patient demographics, clinicopathologic features and immuno-oncologic signatures. Materials-methods: Eighty-seven completely excised OSCC tissues were prospectively collected and scored for histopathologic inflammatory subtypes [HIS]-inflamed (INF), immune-excluded (IE) and immune-desert (ID), peritumoral stromal inflammation (PTSI), and peritumoral stromal fibrosis (PTSF). Scoring of inflammation was complemented by Semaphorin 4D immunohistochemistry. NanoString differential gene expression (DGE) analysis was conducted for eight OSCC cases representative of the inflammatory and stromal subtypes and the demographic groups. Results: PTSF correlated with male gender (p = 0.0043), smoking (p = 0.0455), alcohol consumption (p = 0.0044), increased tumor size (p = 0.0054), and advanced stage (p = 0.002). On the contrary, PTSI occurred predominantly in females (p = 0.0105), non-drinkers (p = 0.0329), and small tumors (p = 0.0044). Transcriptionally, decreased cytokine signaling, and oncogenic pathway activation were observed in HIS-IE. Smokers and males displayed decreased global immune-cell levels and myeloid-cell predominance. Conclusion: Our work describes OSCC stromal and inflammatory phenotypes in correlation with distinct patient groups and DGE, highlighting the translational potential of characterizing the tumor microenvironment for optimal patient stratification.

Tumor microenvironment characteristics association with clinical outcome in patients with resected intestinal-type gastric cancer.

BACKGROUND: Tumor microenvironment (TME) characteristics including tumor stroma ratio (TSR), tumor budding (TB), and tumor-infiltrating lymphocytes (TILs) were examined in resected gastric cancer. These TME features have been shown to indicate metastatic potential in colon cancer, and intestinal-type gastric cancer (IGC) has pathological similarities with that malignancy. METHODS: TSR, TB, and TILs were quantified in routine histological sections from 493 patients with IGC who underwent radical resection at 2 university hospitals in China from 2010 to 2016. TME variables were dichotomized as follows: TSR (50%), TILs (median), TB per international guidelines (4 buds/0.785mm2), and platelet-lymphocyte ratio (PLR) per survival ROC. Association of TME features with patient clinicopathological characteristics, time-to-recurrence (TTR), and cancer-specific-survival (CSS) were examined using univariate and multivariate analysis, including a relative contribution analysis by Cox regression. RESULTS: Patients whose tumors showed high TSR or high TB or low TILs were each significantly associated with increased T and N stage, higher histological grade, and poorer TTR and CSS at 5 years. Only TSR and N stage were independently associated with TTR and CSS after adjustment for covariates. PLR was only independently associated with TTR after adjustment for covariates. Among the variables examined, only TSR was significantly associated with both TTR (HR 1.72, 95% CI, 1.14-2.60, P = .01) and CSS (HR 1.62, 95% CI, 1.05-2.51, P = .03) multivariately. Relative contribution to TTR revealed that the top 3 contributors were N stage (45.1%), TSR (22.5%), and PLR (12.9%), while the top 3 contributors to CSS were N stage (59.9%), TSR (14.7%), and PLR (10.9%). CONCLUSIONS: Among the examined TME features, TSR was the most robust for prognostication and was significantly associated with both TTR and CSS. Furthermore, the relative contribution of TSR to patient TTR and CSS was second only to nodal status.

Tumor-stroma contact ratio - a novel predictive factor for tumor response to chemoradiotherapy in locally advanced oropharyngeal cancer.

The growth pattern of oropharyngeal squamous cell carcinomas (OPSCC) varies from compact tumor cell aggregates to diffusely infiltrating tumor cell-clusters. The influence of the growth pattern on local tumor control and survival has been studied mainly for surgically treated oral cavity carcinomas on a visual basis. In this study, we used multiplex immunofluorescence staining (mIF) to examine the antigens pan-cytokeratin, p16INK4a, Ki67, CD271, PD-L1, and CD8 in pretherapeutic biopsies from 86 OPSCC. We introduce Tumor-stroma contact ratio (TSC), a novel parameter, to quantify the relationship between tumor cells in contact with the stromal surface and the total number of epithelial tumor cells. mIF tumor cores were analyzed at the single-cell level, and tumor-stromal contact area was quantified using the R package "Spatstat". TSC was correlated with the visually assessed invasion pattern by two independent investigators. Furthermore, TSC was analyzed in relation to clinical parameters and patient survival data to evaluate its potential prognostic significance. Higher TSC correlated with poor response to (chemo-)radiotherapy (r = 0.3, p < 0.01), and shorter overall (OS) and progression-free (PFS) survival (median OS: 13 vs 136 months, p < 0.0001; median PFS: 5 vs 85 months, p < 0.0001). Visual categorization of growth pattern according to established criteria of tumor aggressiveness showed interobserver variability increasing with more nuanced categories (2 categories: k = 0.7, 95 %-CI: 0.55 - 0.85; 4 categories k = 0.48, 95 %-CI: 0.35 - 0.61). In conclusion, TSC is an objective and reproducible computer-based parameter to quantify tumor-stroma contact area. We demonstrate its relevance for the response of oropharyngeal carcinomas to primary (chemo-)radiotherapy.