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Macrophages are an essential component of antitumor activity; however, the role of tumor‐associated macrophages (TAMs) in colorectal cancer (CRC) remains controversial. Here, we elucidated the role of TAMs in CRC progression, especially at the early stage. We assessed the TAM number, phenotype, and distribution in 53 patients with colorectal neoplasia, including intramucosal neoplasia, submucosal invasive colorectal cancer (SM‐CRC), and advanced cancer, using double immunofluorescence for CD68 and CD163. Next, we focused on the invasive front in SM‐CRC and association between TAMs and clinicopathological features including lymph node metastasis, which were evaluated in 87 SM‐CRC clinical specimens. The number of M2 macrophages increased with tumor progression and dynamic changes were observed with respect to the number and phenotype of TAMs at the invasive front, especially at the stage of submucosal invasion. A high M2 macrophage count at the invasive front was correlated with lymphovascular invasion, low histological differentiation, and lymph node metastasis; a low M1 macrophage count at the invasive front was correlated with lymph node metastasis. Furthermore, receiver operating characteristic curve analysis revealed that the M2/M1 ratio was a better predictor of the risk of lymph node metastasis than the pan‐, M1, or M2 macrophage counts at the invasive front. These results suggested that TAMs at the invasive front might play a role in CRC progression, especially at the early stages. Therefore, evaluating the TAM phenotype, number, and distribution may be a potential predictor of metastasis, including lymph node metastasis, and TAMs may be a potential CRC therapeutic target., We identified dynamic changes in the number and phenotype of tumor‐associated macrophages (TAMs) at the invasive front in colorectal cancer (CRC), especially at the stage of submucosal invasion. Furthermore, TAMs at the invasive front may play a role in CRC progression, especially in early stage CRC, ie, M1 macrophages at the invasive front may inhibit CRC progression, while M2 macrophages may promote CRC progression via EMT. Therefore, a marker comprising the phenotype, number, and distribution of TAMs may serve as a potential predictor of metastasis, including lymph node metastasis, and TAMs may be a potential therapeutic target in CRC.

Interaction between tumor cells and stromal cells plays an important role in the growth and metastasis of colon cancer. We previously found that carcinoma‐associated fibroblasts (CAFs) expressed platelet‐derived growth factor receptor‐β (PDGFR‐β) and that PDGFR targeted therapy using imatinib or nilotinib inhibited stromal reaction. Bone marrow‐derived mesenchymal stem cells (MSCs) migrate to tumor stroma and differentiate into CAFs. A novel oral multikinase inhibitor regorafenib inhibits receptor tyrosine kinases expressed on stromal cells (vascular endothelial growth factor receptor 1–3, TIE2, PDGFR‐β, and fibroblast growth factors) and tumor cells (c‐KIT, RET, and BRAF). These molecules are involved in tumor growth, angiogenesis, lymphangiogenesis, and stromal activation. Therefore, we examined whether regorafenib impaired the tumor‐promoting effect of CAFs/MSCs. KM12SM human colon cancer cells alone or KM12SM cells with MSCs were transplanted into the cecal wall of nude mice. Co‐implantation of KM12SM cells with MSCs into the cecal wall of nude mice produced tumors with abundant stromal component and promoted tumor growth and lymph node metastasis. Single treatment with regorafenib inhibited tumor growth and metastasis by inhibiting both tumor cells and stromal reaction. This tumor‐inhibitory effect of regorafenib was more obvious in tumors developed by co‐implanting KM12SM cells with MSCs. Our data suggested that targeting of the tumor microenvironment with regorafenib affected tumor cell–MSC interaction, which in turn inhibited the growth and metastasis of colon cancer.

Malic enzyme 1 (ME1) is a multifunctional protein involved in glycolysis, the citric acid cycle, NADPH production, glutamine metabolism, and lipogenesis. It is overexpressed in various cancers. We examined the expression of ME1 in 119 oral squamous cell carcinomas (OSCCs) using immunohistochemistry. Malic enzyme 1 expression was moderate to strong in 57 (48%) OSCCs and correlated with pT, pN, clinical stage, and histological grade. In 37 cases with prognostic evaluation, moderate to strong ME1 expression indicated a worse prognosis than did weak ME1 expression. Malic enzyme 1 knockdown or inactivation by lanthanide inhibited cell proliferation and motility and suppressed the epithelial-mesenchymal transition in HSC3 human OSCC cells. Knockdown of ME1 also shifted energy metabolism from aerobic glycolysis and lactate fermentation to mitochondrial oxidative phosphorylation, and the redox status from reductive to oxidative. In a mouse tumor model, lanthanide suppressed tumor growth and increased survival time. These findings reveal that ME1 is a valid target for molecular therapy in OSCC.

The aim of this study was to clarify predictive factors for response to eradication therapy in cases of Helicobacter pylori (H. pylori)-positive API2-MALT1-negative gastric mucosa-associated lymphoid tissue (MALT) lymphoma. Sixty-six patients who were examined for H. pylori infection and the presence of the API2-MALT1 chimeric transcript and who underwent H. pylori eradication therapy as first-line therapy, were enrolled in this study. Immunohistochemical markers (p53, Ki-67, and BCL10), microsatellite instability, loss of heterozygosity, serum levels of antibodies (anti-H. pylori and anti-CagA), and markers for gastritis (gastrin and pepsinogens) were examined, and the results were compared between patients whose tumors regressed completely after eradication therapy (responders) and patients whose tumors did not regress (non-responders). Of the 66 patients with localized gastric MALT lymphoma, 47 (71.2%) showed complete remission after eradication therapy. None of the H. pylori-negative (n = 9) and/or API2-MALT1-positive (n = 7) patients responded to antibacterial treatment. Of 44 patients with H. pylori-positive API2-MALT1-negative gastric MALT lymphoma, 38 (86.4%) showed complete remission after eradication therapy. Titers of antibodies against H. pylori and CagA protein were significantly higher in the responders than in the non-responders (P = 0.0235 and 0.0089, respectively). No significant difference between the groups was observed for the other factors. In conclusion, measurement of titers of serum antibodies to H. pylori and CagA protein may be useful for predicting the response to eradication therapy in patients with H. pylori-positive API2-MALT1-negative gastric MALT lymphoma.

Primary gastric diffuse large B-cell lymphomas are generally well controlled by non-surgical treatment with combination chemotherapy followed by radiotherapy. We have previously reported that over 90% of patients achieved complete response (CR) with this therapeutic strategy: three cycles of cyclophosphamide, adriamycin, vincristine and prednisone followed by radiotherapy (40.5 Gy). Although the CR rate was very high, some patients still showed resistance to this combination therapy. In order to clarify the factors related to therapy resistance, we examined the relationship between Epstein-Barr virus (EBV), which was examined using an in situ hybridization technique, and the patients' clinical courses. Out of the 50 patients, four were EBV positive; over half of lymphoma cells were positive for EBV by in situ hybridization. Of the three EBV-positive patients, two showed progressive disease and one achieved partial response (PR). Two of the patients died of disease progression. The other patient achieved CR, but the lymphoma recurred with distant metastasis in the cerebellum 3 months after remission. In the present study, eight patients did not achieve CR or they relapsed, four patients showed progressive disease, one patient achieved PR, and three patients achieved CR with recurrence. Therefore, half of these unfavorable patients were EBV positive. This finding strongly indicated that EBV-associated gastric diffuse large B-cell lymphomas frequently show resistance to standard chemoradiotherapy, although some other adverse factors remain unclear.

Vascular endothelial growth factor (VEGF)-C and VEGF-D are potent lymphangiogenic factors produced by tumor and stromal cells. The purpose of this study was to determine whether expression of VEGF-C and/or VEGF-D correlates with clinicopathological features of human colorectal carcinoma. Expression of mRNAs for VEGF-C, VEGF-D, and their receptor VEGFR-3 was examined by reverse transcription-polymerase chain reaction (RT-PCR) in six colon carcinoma cell lines and in fresh endoscopic biopsy specimens from 20 patients with colorectal carcinoma. Expression of VEGF-C and VEGF-D protein was also examined immunohistochemically in 139 archival surgical specimens of human colorectal carcinoma. Of the six cell lines, one (Colo320D) constitutively expressed VEGF-C and four (Colo320D, DLD-1, km12sm, km12c) constitutively expressed VEGF-D mRNA. Expression of VEGF-D mRNA was increased under low oxygen conditions, and all six cell lines constitutively expressed VEGF-D mRNA under hypoxic conditions. Of the 139 specimens of human colorectal carcinoma, 65 (46.8%) showed intense VEGF-C immunoreactivity and 41 (29.5%) showed intense VEGF-D immunoreactivity. In 49 (75.3%) of the 65 and 20 (48.8%) of the 41 cases, heterogeneous intratumoral staining was observed for VEGF-C and VEGF-D, respectively, with the highest levels of expression at the invasive edges. VEGF-C expression correlated with the depth of tumor invasion, lymphatic involvement, venous involvement, lymph node metastasis, and liver metastasis, and VEGF-D expression correlated with the depth of tumor invasion, lymph node metastasis, and liver metastasis. No correlation was observed between VEGF-C and VEGF-D expression in tumors. The survival time of patients with VEGF-C-positive tumors was significantly shorter than that of patients with VEGF-C-negative tumors, and the survival time of patients with VEGF-D-positive tumors was significantly shorter than that of patients with VEGF-D-negative tumors. The survival time of patients with both VEGF-C- and VEGF-D-positive tumors was significantly shorter than that of patients with both VEGF-C- and VEGF-D-negative tumors. These results suggest that VEGF-C and VEGF-D may be independent and important prognostic factors in patients with human colorectal carcinoma.

We conducted a multicenter, retrospective study to determine the anatomical distribution and prognostic factors of gastrointestinal (GI) follicular lymphoma (FL). This study included 125 patients with stage I and II(1) GI-FL. Of the 125 patients, the small intestine was examined in 70 patients, with double-balloon endoscopy and/or capsule endoscopy. The most frequently involved GI-FL site was the duodenal second portion (DSP) (81%), followed by the jejunum (40%); 85% of patients with involvement of the DSP also had jejunal or ileal lesions. The absence of abdominal symptoms and macroscopic appearance of multiple nodules were significantly present in the DSP-positive group. During a median follow up of 40 months, six patients showed disease progression. Patients with involvement of the DSP had better progression-free survival (PFS) than those without such involvement (P = 0.001). A multivariate analysis revealed that male sex, the presence of abdominal symptoms, and negative involvement of the DSP were independently associated with poor PFS. In conclusion, most patients with GI-FL have duodenal lesions associated with multiple jejunal or ileal lesions. Gastrointestinal follicular lymphomas involving the DSP might be a distinct entity showing a favorable clinical course.

Vascular endothelial growth factor (VEGF)-D induces lymphangiogenesis by activating VEGF receptor (VEGFR)-3, which is expressed mainly by lymphatic endothelial cells. VEGFR-3 has also been detected in several types of malignant cells, but the significance of VEGFR-3 expression by malignant cells remains unclear. We examined the expression and function of VEGF-D/VEGFR-3 in human gastric carcinoma cells. Expression of VEGF-D and VEGFR-3 was analyzed in three human gastric carcinoma cell lines and 29 surgical specimens. cDNA microarray analysis was used to examine the effect of VEGF-D on the expression of genes associated with disease progression in VEGFR-3-expressing KKLS cells. VEGF-D-transfected cells and control cells were transplanted into the gastric wall of nude mice. In 10 of the 29 (34%) gastric carcinoma specimens and two of the three cell lines, cancer cells expressed both VEGF-D and VEGFR-3. In vitro treatment of KKLS cells with exogenous VEGF-D increased expression of cyclin D1 and Bcl-2 and stimulated cell proliferation. VEGF-D transfection into KKLS cells resulted in stimulation of angiogenesis, lymphangiogenesis, and cell proliferation, and in inhibition of apoptosis. VEGF-D may participate in the progression of human gastric carcinoma by acting via autocrine and paracrine mechanisms.

Recent study of murine fibrosarcoma has revealed that platelet-derived growth factor (PDGF) plays a direct role in promoting lymphangiogenesis and metastatic spread to lymph nodes. Thus, we investigated the relation between PDGF and PDGF receptor (PDGF-R) expression and lymphatic metastasis in human gastric carcinoma. We examined PDGF-B and PDGF-Rβ expression in four human gastric carcinoma cell lines (TMK-1, MKN-1, MKN-45, and KKLS) and in 38 surgical specimens of gastric carcinoma. PDGF-B and PDGF-Rβ expression was examined by immunofluorescence in surgical specimens and in human gastric carcinoma cells (TMK-1) implanted orthotopically in nude mice. Groups of mice (n = 10, each) received saline (control) or PDGF-R tyrosine kinase inhibitor imatinib. PDGF-B and PDGF-Rβ mRNA expression was significantly higher in patients with lymph node metastasis than in those without and was also significantly higher in diffuse-type carcinoma than in intestinal-type carcinoma. In surgical specimens, tumor cells expressed PDGF-B, but PDGF-Rβ was expressed predominantly by stromal cells. Under culture conditions, expression of PDGF-B mRNA was found in all of the gastric cell lines, albeit at different levels. In orthotopic TMK-1 tumors, cancer cells expressed PDGF-B but not PDGF-Rβ. PDGF-Rβ was expressed by stromal cells, including lymphatic endothelial cells. Four weeks of treatment with imatinib significantly decreased the area of lymphatic vessels. Our data indicate that secretion of PDGF-B by gastric carcinoma cells and expression of PDGF-Rβ by tumor-associated stromal cells are associated with lymphatic metastasis. Blockade of PDGF-R signaling pathways may inhibit lymph node metastasis of gastric carcinoma.

We screened for mutations in the von Hippel-Lindau (VHL) tumor suppressor gene and examined the relationship of these mutations with expression of hypoxia-inducible factor (HIF)-1alpha protein in human colorectal carcinomas. DNAs were extracted from 130 paraffin-embedded tumor specimens and subjected to polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) analysis followed by direct sequencing. We identified 13 mutations in the coding sequence of VHL, 12 of which were unique events. Three mutations were located in exon 2 and the others in exon 3. These mutations were detected in 10 of 88 (11.4%) tumors tested. Furthermore, seven of the 13 (53.8%) VHL mutants immunohistochemically showed high HIF-1alpha expression. The mean percentage of cells with strong cytoplasmic HIF-1alpha expression was 67.5% in tumors with VHL mutations, and this level was significantly higher than that in tumors without mutations (50.8%, P < 0.05). These findings suggest that mutations in VHL may play a role in colorectal carcinoma via activation of the HIF-related transcriptional cascade.