Prognostic value of polarized macrophages in patients with hepatocellular carcinoma after curative resection

As the most predominant tumour‐infiltrating immune cells, tumour‐associated macrophages (TAMs) are significant for fostering tumour growth, progression and metastasis. CD68‐positive TAMs display dissimilarly polarized programmes comprising CD11c‐positive pro‐inflammatory macrophages (M1) and CD206‐positive immunosuppressive macrophages (M2). The aim of this study is to determine the prognostic significance of diametrically polarized TAMs in hepatocellular carcinoma (HCC) and their application to risk stratification of patients according to their specific prognostic values. This study included 80 consecutive patients with HCC, and we evaluated diametrically polarized functional status of macrophages by immunohistochemical staining of CD68, CD11c and CD206. Prognostic values and clinicopathologic features were assessed in these patients. High versus low CD11c‐positive TAM density (P = 0.005) and low versus high CD206‐positive TAM density (P = 0.002) were associated with better overall survival, whereas CD68‐positive TAM density had no prognostic significance (low versus high, P = 0.065). Furthermore, the presence of these positive staining macrophages did not show any prognostic significance for recurrence‐free survival (all P > 0.05). Multivariate Cox regression analysis identified CD11c‐positive and CD206‐positive TAMs as an independent prognostic factor (P < 0.001, P = 0.031, respectively). Intratumoural infiltration of diametrically polarized TAMs, a novel identified independent prognostic factor for survival in patients with HCC, could be combined with the TNM stage and the Barcelona Clinic Liver Cancer stage to improve a risk stratification system.     

The correlation between tumor-associated macrophage infiltration and progression in cervical carcinoma

Tumor microenvironment (TME) plays a particularly important role in the progression, invasion and metastasis of cervical carcinoma (CC). Tumor-associated macrophages (TAMs) are significant components of the tumor microenvironment in CC. However, the results of studies on the correlation between TAMs and progression in CC are still controversial. This research aimed to investigate the relationship between TAMs infiltration and progression in CC. A total of 100 patients with CC were included in the study. The correlation between TAMs and clinicopathologic features was studied. Besides, a systematic literature search was conducted from legitimate electronic databases to specifically evaluate the role of TAMs in TME of cervical carcinoma. In the meta-analysis, high stromal CD68⁺ TAMs density was relevant to lymph node metastasis (WMD = 11.89, 95% CI: 5.30–18.47). At the same time, CD163⁺ M2 TAM density was associated with lymph node metastasis (OR = 2.42, 95% CI: 1.09–5.37; WMD = 39.37, 95% CI: 28.25–50.49) and FIGO stage (WMD = -33.60, 95% CI: -45.04 to -22.16). This was further confirmed in the experimental study of 100 tissues of cervical cancer. It supported a critical role of TAMs as a prospective predictor of cervical cancer. In conclusion, CD68⁺ TAM and CD163⁺ M2 TAM infiltration in CC were associated with tumor progression. And CD163⁺ M2 TAM infiltration was associated with more advanced FIGO stage and lymph node metastasis in CC.     

The Clinical Significance of the CD163+ and CD68+ Macrophages in Patients with Hepatocellular Carcinoma

Our previous study has found that the abundance of peritumoral CD68⁺ macrophages was associated with poor prognosis in hepatocellular carcinoma (HCC) after resection. However, CD68 staining could not discriminate the protumoral or tumoricidal subpopulations from pan-macrophages. CD163 is a marker of alternatively activated macrophages. In this study, the clinical significance of CD163⁺ cells in tumors and peritumoral liver tissues was evaluated in a cohort of 295 patients with HCC after curative resection. We found that the density of CD163⁺ cells was well correlated with that of CD68⁺ cells in both tumors and peritumoral liver tissues but was much more. Immunostaining on consecutive sections and flow cytometry assay on surgical resected specimens further supported the findings that the CD163⁺ cells was more abundant than CD68⁺ cells. The density of peritumoral CD68⁺ cells was associated with poor recurrence-free survival (RFS) and poor overall survival (OS) (P = 0.004 and P = 0.001, respectively), whereas the CD163⁺ cells have no prognostic values either in tumors or in peritumoral liver tissues. In another cohort of 107 HCC patients, preoperative plasma concentration of soluble form of CD163 (sCD163) was associated with active hepatitis-related factors but not associated with the markers of tumor invasion. In conclusion, both the CD163⁺ cells local infiltration and plasma sCD163 were of limited significance in HCC, and they were more likely markers related to active hepatitis rather than tumor progression.     

High MUC2 Expression in Ovarian Cancer Is Inversely Associated with the M1/M2 Ratio of Tumor-Associated Macrophages and Patient Survival Time

Mucin 2 (MUC2) is a mucin molecule aberrantly expressed by ovarian cancer cells. Previous in vitro studies have indicated that MUC2 promotes cancer growth and metastasis through a tumor-associated macrophage (TAM)-dependent mechanism. However, this mechanism has never been linked to clinical oncology, and its prognostic significance needed to be clarified. Here, we collected 102 consecutive ovarian cancer specimens and used the multiple immuno-histo-chemical/-fluorescent technique to determine the correlations between the MUC2 expression status, the ratio of M1/M2 TAMs and the densities of cyclooxygenase-2 (COX-2)⁺ TAMs and COX-2⁺ cancer cells. The Kaplan-Meier survival analysis and multivariate Cox regression analysis were used to evaluate the prognostic influences of these parameters. As a result, we found that the MUC2 overexpression (immunostaining ++/+++) was significantly correlated with a reduced ratio of M1/M2 TAMs (p<0.001), an increased density of COX-2⁺ TAMs (p<0.001) and an increased density of COX-2⁺ cancer cells (p=0.017). Moreover, most of the M2 TAMs (93%-100%) and COX-2⁺ TAMs (63%-89%) overlapped; and the COX-2⁺ cancer cells were frequently observed near the COX-2⁺ TAMs. In the Cox regression analysis, MUC2 overexpression was found to be an independent prognostic factor for ovarian cancer patients, of which the hazard ratio (HR) was 2.354 (95% confidence interval (CI): 1.031-10.707, p=0.005). Also, the reduced ratio of M1/M2 TAMs and the increased densities of COX-2⁺ TAMs and COX-2⁺ cancer cells were demonstrated to be the predictors of poor prognosis, among which the reduced M1/M2 ratio possessed the highest HR (1.767, 95% CI: 1.061-6.957, p=0.019). All these findings revealed that MUC2 can concurrently exert M2-polarizing and COX-2-inducing effects on TAMs, by which it causes an imbalanced TAM M1-/M2-polarization pattern and induces local PGE₂ synthesis (in both TAMs and cancer cells). The positive feedback between local PGE₂ synthesis and TAM M2-polarization accelerates ovarian cancer progression.     

Foxp3+ cell infiltration and granzyme B+/Foxp3+ cell ratio are associated with outcome in neoadjuvant chemotherapy-treated ovarian carcinoma

Preoperative neoadjuvant chemotherapy (NAC) can significantly reduce tumour burden in patients with primarily unresectable chemosensitive tumours, allowing a more complete cytoreduction during debulking surgery and facilitating evaluation of tumour chemosensitivity, identification of appropriate treatment options and improvement of intervention protocols. In this study, we investigate, using immunohistochemistry, the impact of platinum/taxane-based NAC (NAC) on tumour-infiltrating lymphocytes (TILs) in advanced epithelial ovarian cancer (EOC) and their relationship with clinical outcome. All patients had clinical response, as shown by ascites volume and CA125 levels compared to pre-treatment findings. NAC intervention significantly increased CD4⁺, CD8⁺ and granzyme B⁺ infiltration while Foxp3⁺ accumulation remained unaffected. TILs were prognostically neutral for both progression-free survival (PFS) and overall survival (OS) before NAC. In contrast, after NAC, elevated granzyme B⁺ infiltration displayed a tendency for improved PFS (log-rank 0.064). Further, low Foxp3⁺ cell density was associated with longer PFS, as compared with strong Foxp3⁺ infiltration (median 20.94 vs. 11.24 months; log-rank 0.0001) and with improved OS (median 30.75 vs. 16.04 months, respectively; log-rank 0.056), demonstrating clear prognostic significance for PFS. In addition, high granzyme B⁺/Foxp3⁺ ratio post-NAC strongly correlated with improved PFS compared to low granzyme B⁺/Foxp3⁺ cell ratio (median 17.88 vs. 11.24 months, respectively), and showed to be a favourable prognostic factor for PFS (log-rank 0.014). Our findings indicate that NAC elicited an immunologic profile in which low immunosuppressive Foxp3⁺ infiltration and elevated numbers of activated granzyme B⁺ cells were significantly associated with EOC-specific PFS, suggesting a contribution of immunologic effects to improved clinical outcome.     

The presence of LC3B puncta and HMGB1 expression in malignant cells correlate with the immune infiltrate in breast cancer

Several cell-intrinsic alterations have poor prognostic features in human breast cancer, as exemplified by the absence of MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3 β)-positive puncta in the cytoplasm (which indicates reduced autophagic flux) or the loss of nuclear HMGB1 expression by malignant cells. It is well established that breast cancer is under strong immunosurveillance, as reflected by the fact that scarce infiltration of the malignant lesion by CD8⁺ cytotoxic T lymphocytes or comparatively dense infiltration by immunosuppressive cell types (such as FOXP3⁺ regulatory T cells or CD68⁺ tumor-associated macrophages), resulting in low CD8⁺:FOXP3⁺ or CD8⁺:CD68⁺ ratios, has a negative prognostic impact. Here, we reveal the surprising finding that cell-intrinsic features may influence the composition of the immune infiltrate in human breast cancer. Thus, the absence of LC3B puncta is correlated with intratumoral (but not peritumoral) infiltration by fewer CD8⁺ cells and more FOXP3⁺ or CD68⁺ cells, resulting in a major drop in the CD8⁺:FOXP3⁺ or CD8⁺:CD68⁺ ratios. Moreover, absence of HMGB1 expression in nuclei correlated with a general drop in all immune effectors, in particular FOXP3⁺ and CD68⁺ cells, both within the tumor and close to it. Combined analysis of LC3B puncta and HMGB1 expression allowed for improved stratification of patients with respect to the characteristics of their immune infiltrate as well as overall and metastasis-free survival. It can be speculated that blocked autophagy in, or HMGB1 loss from, cancer cells may favor tumor progression due to their negative impact on anticancer immunosurveillance.     

Prognostic significance of combining high mobility group Box-1 and OV-6 expression in hepatocellular carcinoma

The inflammatory environment and existence of cancer stem cells are critical for progression and intrahepatic recurrence of hepatocellular carcinoma (HCC) after curative resections. Here, we investigated the prognostic significance of combining high mobility group box 1 (HMGB1) expression and hepatic progenitor marker OV6 in hepatocellular carcinoma. Expression of HMGB1 and OV6 was evaluated using immunohistochemistry profiling in tissue microarrays containing samples from 208 HCC patients. Invasive clinical or pathological factors were found in patients with high expression of HMGB1 or OV6. Higher HMGB1 was associated with poorer clinical outcomes, and independently related to elevated 5-year recurrence incidence (85.5% vs. 62.4%, P<0.001). We also found that more OV6 positive staining was correlated with poor prognosis of HCC patients (P<0.001). Notably, expression of HMGB1 was positively correlated with OV6 in density (R²=0.032, P<0.001) and reversely related to HCC outcomes. Abnormal expression of HMGB1 in combination with positive staining of OV6 displayed poorer prognostic performance than single biomarker alone (area under curve (AUC) survival=0.696). Therefore, HMGB1 and OV6 positive staining are promising prognostic parameters for HCC, and we propose that HMGB1 and OV6 may cooperate with each other and predict poor prognosis of HCC.     

Rejection of intradermally injected syngeneic tumor cells from mice by specific elimination of tumor-associated macrophages with liposome-encapsulated dichloromethylene diphosphonate, followed by induction of CD11b+/CCR3−/Gr-1− cells cytotoxic against the tumor cells

Tumor cell expansion relies on nutrient supply, and oxygen limitation is central in controlling neovascularization and tumor spread. Monocytes infiltrate into tumors from the circulation along defined chemotactic gradients, differentiate into tumor-associated macrophages (TAMs), and then accumulate in the hypoxic areas. Elevated TAM density in some regions or overall TAM numbers are correlated with increased tumor angiogenesis and a reduced host survival in the case of various types of tumors. To evaluate the role of TAMs in tumor growth, we here specifically eliminated TAMs by in vivo application of dichloromethylene diphosphonate (DMDP)-containing liposomes to mice bearing various types of tumors (e.g., B16 melanoma, KLN205 squamous cell carcinoma, and 3LL Lewis lung cancer), all of which grew in the dermis of syngeneic mouse skin. When DMDP-liposomes were injected into four spots to surround the tumor on day 0 or 5 after tumor injection and every third day thereafter, both the induction of TAMs and the tumor growth were suppressed in a dose-dependent and injection number-dependent manner; and unexpectedly, the tumor cells were rejected by 12 injections of three times-diluted DMDP-liposomes. The absence of TAMs in turn induced the invasion of inflammatory cells into or around the tumors; and the major population of effector cells cytotoxic against the target tumor cells were CD11b⁺ monocytic macrophages, but not CCR3⁺ eosinophils or Gr-1⁺ neutrophils. These results indicate that both the absence of TAMs and invasion of CD11b⁺ monocytic macrophages resulted in the tumor rejection.     

The Prognostic Implications of Macrophages Expressing Proliferating Cell Nuclear Antigen in Breast Cancer Depend on Immune Context

Tumor associated macrophages (TAMs) are recruited from the circulation to the tumor site, and can undergo a spectrum of phenotypic changes, with two contrasting activation states described in the literature: the M1 and M2 phenotypes. We previously identified a population of TAMs that express proliferating cell nuclear antigen (PCNA) and are associated with high grade, hormone receptor negative breast cancers and poor outcomes. In the present exploratory study we again found that high PCNA⁺ TAM counts in pre-treatment tumor biopsies (102 invasive breast cancer cases from the I-SPY 1 Trial, a prospective neoadjuvant trial with serial core biopsies and gene array data) were associated with high grade, hormone receptor negativity, and decreased recurrence free survival. We explored the association of these PCNA⁺ TAMs with the expression of M1 and M2 related genes and, contrary to expectation, observed that high PCNA⁺ TAM levels were associated with more M1- than M2-related genes. An immune gene signature, derived from cytotoxic T cell and MHC Class II genes (Tc/ClassII), was developed and we found that high PCNA⁺ TAM counts, in the context of a low Tc/ClassII signature score, were associated with significantly worse recurrence free survival in all cases and in hormone receptor negative only cases. We observed similar results using a gene signature-proxy for PCNA⁺ TAMs in a larger independent set of 425 neoadjuvant-treated breast cancer cases. The results of this exploratory study indicate that high numbers of PCNA⁺ TAMs, in the absence of an anti-tumor immune microenvironment (as indicated by a low Tc/ClassII signature score), are associated with poor outcomes in breast cancer patients treated with neoadjuvant chemotherapy. This, along with the observation that PCNA⁺ TAMs were associated predominantly with M1-related genes, may provide new insights into the role of the immune microenvironment in breast cancer.     

CXCL17 Expression Predicts Poor Prognosis and Correlates with Adverse Immune Infiltration in Hepatocellular Carcinoma

CXC ligand 17 (CXCL17) is a novel CXC chemokine whose clinical significance remains largely unknown. In the present study, we characterized the prognostic value of CXCL17 in patients with hepatocellular carcinoma (HCC) and evaluated the association of CXCL17 with immune infiltration. We examined CXCL17 expression in 227 HCC tissue specimens by immunohistochemical staining, and correlated CXCL17 expression patterns with clinicopathological features, prognosis, and immune infiltrate density (CD4 T cells, CD8 T cells, B cells, natural killer cells, neutrophils, macrophages). Kaplan-Meier survival analysis showed that both increased intratumoral CXCL17 (P = 0.015 for overall survival [OS], P = 0.003 for recurrence-free survival [RFS]) and peritumoral CXCL17 (P = 0.002 for OS, P<0.001 for RFS) were associated with shorter OS and RFS. Patients in the CXCL17^low group had significantly lower 5-year recurrence rate compared with patients in the CXCL17^high group (peritumoral: 53.1% vs. 77.7%, P<0.001, intratumoral: 58.6% vs. 73.0%, P = 0.001, respectively). Multivariate Cox proportional hazards analysis identified peritumoral CXCL17 as an independent prognostic factor for both OS (hazard ratio [HR] = 2.066, 95% confidence interval [CI] = 1.296–3.292, P = 0.002) and RFS (HR = 1.844, 95% CI = 1.218–2.793, P = 0.004). Moreover, CXCL17 expression was associated with more CD68 and less CD4 cell infiltration (both P<0.05). The combination of CXCL17 density and immune infiltration could be used to further classify patients into subsets with different prognosis for RFS. Our results provide the first evidence that tumor-infiltrating CXCL17⁺ cell density is an independent prognostic factor that predicts both OS and RFS in HCC. CXCL17 production correlated with adverse immune infiltration and might be an important target for anti-HCC therapies.     

Decreased expression of interleukin-36α correlates with poor prognosis in hepatocellular carcinoma

Interleukin-36α (IL-36α) has been found to have a prominent role in the pathogenesis of inflammatory disorders; however, little is known about the role of IL-36α in cancer. In this study, we investigated the expression, prognostic value, and the underlying antitumor mechanism of IL-36α in hepatocellular carcinoma (HCC). From immunohistochemistry analysis, IL-36α expression was lower in poorly differentiated HCC cells. In clinicopathological analysis, low IL-36α expression significantly correlated with tumor size, histological differentiation, tumor stage, and vascular invasion, and low intratumoral IL-36α expression had significantly worse overall survival rates and shorter disease-free survival rates. Moreover, intratumoral IL-36α expression was an independent risk factor for overall survival. Consecutive sections were used to detect CD3⁺, CD8⁺, and CD4⁺ tumor-infiltrating lymphocytes (TILs), and we found that high-IL-36α-expressing tumor tissues exhibited a significantly higher proportion of intratumoral CD3⁺ and CD8⁺ TILs, but not CD4⁺ TILs. Our in vitro model confirmed that supernatant from IL-36α-overexpressing human HCC cells had an increased capacity to recruit CD3⁺ and CD8⁺ T cells. Consistently, mouse HCC cells engineered to overexpress IL-36α demonstrated markedly delayed growth in vivo, as well as higher levels of intratumoral CD3⁺ and CD8⁺ TILs, compared with control mice. In vitro chemotaxis analysis also showed that mouse HCC cells overexpressing IL-36α could recruit more number of CD3⁺ and CD8⁺ T cells. These results show that IL-36α expression may play a pivotal role in determining the prognosis of patients with HCC, which we attribute to the activation of adaptive T cell immunity, especially CD8⁺ T cell immune response.     

Prognostic Value of Tumor-Associated Macrophages According to Histologic Locations and Hormone Receptor Status in Breast Cancer

Tumor-associated macrophages (TAMs) are involved in tumor progression by promoting epithelial-mesenchymal transition (EMT), tumor cell invasion, migration and angiogenesis. However, in breast cancer, the clinical relevance of the TAM infiltration according to distinct histologic locations (intratumoral vs. stromal) and hormone receptor status is unclear. We investigated the significance of the levels of TAM infiltration in distinct histologic locations in invasive breast cancer. We also examined the relationship of the TAM levels with the clinicopathologic features of tumors, expression of EMT markers, and clinical outcomes. Finally, we analyzed the prognostic value of TAM levels according to hormone receptor status. High levels of infiltration of intratumoral, stromal and total TAMs were associated with high histologic grade, p53 overexpression, high Ki-67 proliferation index and negative hormone receptor status. Infiltration of TAMs was also correlated with overexpression of vimentin, smooth muscle actin and alteration of β-catenin. Overall, a high level of infiltration of intratumoral TAMs was associated with poor disease-free survival, and was found to be an independent prognostic factor. In subgroup analyses by hormone receptor status, a high level of infiltration of intratumoral TAM was an independent prognostic factor in the hormone receptor-positive subgroup, but not in the hormone-receptor negative subgroup. Our findings suggest that intratumoral TAMs play an important role in tumor progression in breast cancer, especially in the hormone receptor-positive group, and the level of TAM infiltration may be used as a prognostic factor and even a therapeutic target in breast cancer.     

High density of IL-17-producing cells is associated with improved prognosis for advanced epithelial ovarian cancer

Interleukin (IL)-17 is the signature cytokine of T helper 17 cells. The role of IL-17 in the tumor microenvironment is still controversial. Few studies describing IL-17 expression in ovarian cancer have been reported. We have therefore analyzed the in situ tumor expression of IL-17 in advanced ovarian cancer and the possible correlation of IL-17 expression with tumor-associated macrophages (TAMs) and with survival in advanced ovarian cancer. Clinical data of 104 patients with stage III–IV epithelial ovarian cancer at the Sun Yat-sen University Cancer Center between 2000 and 2008 were retrospectively reviewed. Immunohistochemical staining of IL-17 and CD163 (marker for TAMs) was performed. Our data showed that levels of IL-17 were significantly increased in ovarian cancer compared with normal ovarian tissues (P<0.001). The high IL-17 expression group included more patients with grade 1 tumors than the low IL-17 expression group (P=0.042). High IL-17 expression correlated with improved progression-free survival (PFS) in advanced ovarian cancer (P<0.001). However, no significant difference was observed in overall survival between the high and low IL-17 expression groups. Multivariate analysis revealed that the density of IL-17-producing cells was a positive prognostic factor for PFS (P=0.001). Moreover, a positive correlation between the density of IL-17-producing cells and TAMs was identified (r=0.354, P<0.001). Our results indicate that the infiltration of IL-17-producing cells might contribute to improved PFS in advanced ovarian cancer. Our findings provide a new insight into the complex role of IL-17 in the tumor microenvironment of ovarian cancer.     

Prognostic Value of Tumor-Infiltrating FoxP3+ T Cells in Gastrointestinal Cancers: A Meta Analysis

Purpose

Tumor-infiltrating FoxP3⁺ T cells have been reported in various human tumors, which impaired cell-mediated immunity and promoted disease progression. However, its prognostic value for survival in patients with different gastrointestinal cancers [hepatocellular carcinoma (HCC), colorectal cancer (CRC), gastric cancer (GC)] remains controversial.

Methods

Relevant literature was searched using PubMed, Embase, Cochrane, Ovid Medline and Chinese wanfang databases. A meta-analysis was conducted to estimate pooled survival and recurrence ratios. The odds ratio (OR) and 95% confidence intervals (CI) were calculated employing fixed- or random-effects models depending on the heterogeneity of the included trials.

Results

For HCC and GC, the overall survival at 1, 3 and 5-year of high FoxP3⁺ T cells infiltration patients were lower than low FoxP3⁺ T cells infiltration patients (P<0.05). The recurrences at 1, 3 and 5-year of high FoxP3⁺ T cells infiltration patients were higher than low FoxP3⁺ T cells infiltration patients (P<0.001). But for CRC, the overall survival at 1, 3 and 5-year of high FoxP3⁺ T cells infiltration patients were higher than low FoxP3⁺ T cells infiltration patients (P<0.001). There were no differences in 1, 3 and 5-year recurrences between high and low FoxP3⁺ T cells infiltration patients (P>0.05).

Conclusions

Our findings suggested that tumor-infiltrating FoxP3⁺ T cells were a factor for a poor prognosis for HCC and GC, but a good prognosis for CRC.     

Dysfunctional Activation of Neurotensin/IL-8 Pathway in Hepatocellular Carcinoma Is Associated with Increased Inflammatory Response in Microenvironment,More Epithelial Mesenchymal Transition in Cancer and Worse Prognosis in Patients

Aim

To investigate the role of neurotensin (NTS) in hepatocellular carcinoma (HCC) sub- grouping and the clinical and pathological significance of activation of NTS/IL-8 pathway in HCC.

Methods

The genome-wide gene expression profiling were conducted in 10 pairs of cancer tissues and corresponding normal adjacent tissues samples using Affymetrix GeneChip® Human Genome U133 Plus 2.0 microarray to screen differentially expressing genes and enrich dysfunctional activated pathways among different HCC subgroups. The levels of NTS protein and multiple inflammation and epithelial mesenchymal transition (EMT) related proteins, including IL-8, VEGF, MMP9, CD68, E-Cadherin, β-Catenin and Vimentin were examined in 64 cases of paraffin-embedded HCC samples using immunohistochemistry (IHC) staining method. The clinical outcome and overall survival (OS) were compared.

Results

A subgroup of HCC characterized by up-regulated NTS expression was accompanied by up-regulated inflammatory responses and EMT. The direct interaction between NTS and IL-8 was identified by pathway enrichment analysis. Significantly increased IL-8 protein was confirmed in 90.91% of NTS⁺ HCC samples and significantly positively correlated to the levels of NTS protein in cancer tissues (P = 0.036), which implied activation of NTS/IL-8 pathway in HCC. The levels of VEGF and MMP9 correlated with co-expression of NTS and IL-8. Increased infiltration of CD68⁺ macrophages and more cancer cells displaying EMT features were found in NTS⁺IL-8⁺ samples. The co-expression of NTS and IL-8 in cancer significantly correlated with the clinical outcomes, as the mortality rate of NTS⁺IL-8⁺ HCC patients is 2.5-fold higher than the others after the surgery (P = 0.022). Accordingly, the OS of NTS⁺IL-8⁺ HCC patients significantly decreased who are under a higher hazard of death at an expected hazard ratio (HR) of 3.457.

Conclusion

Dysfunctional activation of the NTS/IL-8 pathway was detected in HCC which is associated with increased inflammatory response in microenvironment, enhanced EMT in cancer, and worse prognosis in HCC patients.     

Molecular characterization of lung cancer: A two-miRNA prognostic signature based on cancer stem-like cells related genes

Lung cancer is one of the deadliest cancers worldwide. To increase the survival rate of lung cancer, it is necessary to explore specific prognosis markers. More and more evidence finds that noncoding RNA is closely associated with the survival of lung cancer, and cancer stem cells (CSCs) also play a significant role in the progress of lung cancer. The objective of this study is to find CSLCs genes that affect the prognosis of lung cancer. The differential expression of long noncoding RNAs (lncRNAs), microRNAs (miRNAs), messenger RNAs (mRNAs) in the Cancer Genome Atlas (TCGA) database and differential expression data from microarray of CD326⁺ and CD326⁻ A549 cell are intersected to identify stable and consistent expression genes (2 lncRNAs, 15 miRNAs, and 134 mRNAs). The intersection of lncRNAs and miRNAs is analyzed by univariate and multivariate Cox regression to obtained prognostic genes. Two miRNAs (miR-30b-5p and miR-29c-3p) are significantly correlated with the overall survival rate. Then using these two miRNAs to construct a risk score model as a prognosis signature of lung cancer. Subsequently, we analyzed the association between two miRNAs and clinical information of lung cancer patients, of which T stage, Neoplasm cancer and risk score (P < .05) can be used as independent prognostic indicators of lung cancer. Finally, target genes of 2 miRNAs and 134 mRNAs were annotated with Gene Ontology and analyzed with Kyoto Encyclopedia of Genes and Genomes pathway, and verified with the GEO database. In summary, this study illustrates the role of miRNAs in the promotion of lung cancer by CSCs, which is important to find molecular biomarkers of lung cancer.     

The Enhanced Metastatic Potential of Hepatocellular Carcinoma (HCC) Cells with Sorafenib Resistance

Acquired resistance towards sorafenib treatment was found in HCC patients, which results in poor prognosis. To investigate the enhanced metastatic potential of sorafenib resistance cells, sorafenib-resistant (SorR) cell lines were established by long-term exposure of the HCC cells to the maximum tolerated dose of sorafenib. Cell proliferation assay and qPCR of ABC transporter genes (ABCC1-3) were first performed to confirm the resistance of cells. Migration and invasion assays, and immunoblotting analysis on the expression of epithelial to mesenchymal transition (EMT) regulatory proteins were performed to study the metastatic potential of SorR cells. The expression of CD44 and CD133 were studied by flow cytometry and the gene expressions of pluripotency factors were studied by qPCR to demonstrate the enrichment of cancer stem cells (CSCs) in SorR cells. Control (CTL) and SorR cells were also injected orthotopically to the livers of NOD-SCID mice to investigate the development of lung metastasis. Increased expressions of ABCC1-3 were found in SorR cells. Enhanced migratory and invasive abilities of SorR cells were observed. The changes in expression of EMT regulatory proteins demonstrated an activation of the EMT process in SorR cells. Enriched proportion of CD44⁺ and CD44⁺CD133⁺ cells were also observed in SorR cells. All (8/8) mice injected with SorR cells demonstrated lung metastasis whereas only 1/8 mouse injected with CTL cells showed lung metastasis. HCC cells with sorafenib resistance demonstrated a higher metastatic potential, which may be due to the activated EMT process. Enriched CSCs were also demonstrated in the sorafenib resistant cells. This study suggests that advanced HCC patients with acquired sorafenib resistance may have enhanced tumor growth or distant metastasis, which raises the concern of long-term sorafenib treatment in advanced HCC patients who have developed resistance of sorafenib.     

Prognostic Implication of M2 Macrophages Are Determined by the Proportional Balance of Tumor Associated Macrophages and Tumor Infiltrating Lymphocytes in Microsatellite-Unstable Gastric Carcinoma

Tumor associated macrophages are major inflammatory cells that play an important role in the tumor microenvironment. In this study, we investigated the prognostic significance of tumor associated macrophages (TAMs) in MSI-high gastric cancers using immunohistochemistry. CD68 and CD163 were used as markers for total infiltrating macrophages and M2-polarized macrophages, respectively. The density of CD68+ or CD163+ TAMs in four different areas (epithelial and stromal compartments of both the tumor center and invasive front) were analyzed in 143 cases of MSI-high advanced gastric cancers using a computerized image analysis system. Gastric cancers were scored as “0” or “1” in each area when the density of CD68+ and CD163+ TAMs was below or above the median value. Low density of CD68+ or CD163+ macrophages in four combined areas was closely associated with more frequent low-grade histology and the intestinal type tumor of the Lauren classification. In survival analysis, the low density of CD163+ TAMs was significantly associated with poor disease-free survival. In multivariate survival analysis, CD163+ TAMs in four combined areas, stromal and epithelial compartments of both tumor center and invasive front were independent prognostic indicator in MSI-high gastric cancers. In addition, the density of CD163+ TAMs correlated with tumor infiltrating lymphocytes (TILs). Our results indicate that the high density of CD163+ TAMs is an independent prognostic marker heralding prolonged disease-free survival and that the prognostic implication of CD163+ TAMs might be determined by the proportional balance of TAMs and TILs in MSI-high gastric cancers.     

Analysis of the efficacy,safety and survival factors of stereotactic body radiation therapy in patients with recurrence of pancreatic cancer

Objective: This study aims to evaluate the efficacy and safety of stereotactic body radiation therapy (SBRT) using Cyber Knife (CK) in the treatment of patients with recurrent pancreatic cancer after surgery, and analyze its survival-related factors. Methods: The primary endpoint was freedom from local progression (FFLP) and local control (LC) rate after CK. The secondary endpoints were overall survival (OS), progression-free survival (PFS), symptom relief and toxicities. Receiver operating characteristic (ROC) curves were used to determine the optimal cut-off values of inflammatory composite indicators NLR, PLR, SII and PNI. The prognostic factors that affected these patients were analyzed by univariate and multivariate analysis, respectively. Results: A total of 27 patients were enrolled. Median local recurrence disease free interval（DFI）was 11.3 (1.3-30.6) months, LC was 81.5% and 37.0% at 6 and 12 months, respectively. Median PFS was 7.1 (1.3-27.1) months. Median OS was 11.3 (1.3-30.6) months. Symptom alleviation was observed in 16 of 17 patients (94.1%) within 2 weeks after CK. Subsequent chemotherapy, CA199≥50% decrease after CK were independent prognostic factors for OS (all P <0.05). Conclusion: SBRT is a safe and effective treatment approach for recurrent pancreatic adenocarcinoma. Encouraging local control rate, low toxicity, and effective symptom relief suggests the vital role of CK in the treatment of these patients. This clinical application needs to be further studied in the combination of CK and multimodal therapy.