Synergistic antitumor activity by dual blockade of CCR1 and CXCR2 expressed on myeloid cells within the tumor microenvironment.

BACKGROUND: Chemokine signaling within the tumor microenvironment can promote tumor progression. Although CCR1 and CXCR2 on myeloid cells could be involved in tumor progression, it remains elusive what effect would be observed if both of those are blocked. METHODS: We employed two syngeneic colorectal cancer mouse models: a transplanted tumor model and a liver metastasis model. We generated double-knockout mice for CCR1 and CXCR2, and performed bone marrow (BM) transfer experiments in which sub-lethally irradiated wild-type mice were reconstituted with BM from either wild-type, Ccr1-/-, Cxcr2-/- or Ccr1-/-Cxcr2-/- mice. RESULTS: Myeloid cells that express MMP2, MMP9 and VEGF were accumulated around both types of tumors through CCR1- and CXCR2-mediated pathways. Mice reconstituted with Ccr1-/-Cxcr2-/- BM exhibited the strongest suppression of tumor growth and liver metastasis compared with other three groups. Depletion of CCR1+CXCR2+ myeloid cells led to a higher frequency of CD8+ T cells, whereas the numbers of Ly6G+ neutrophils, FOXP3+ Treg cells and CD31+ endothelial cells were significantly decreased. Furthermore, treatment with a neutralizing anti-CCR1 mAb to mice reconstituted with Cxcr2-/- BM significantly suppressed tumor growth and liver metastasis. CONCLUSION: Dual blockade of CCR1 and CXCR2 pathways in myeloid cells could be an effective therapy against colorectal cancer.

Downregulation of osteoprotegerin in colorectal cancer cells promotes liver metastasis via activating tumor-associated macrophage.

Osteoprotegerin (OPG) is a secreted cytokine that functions as a decoy receptor for receptor activator of nuclear factor kappa-B (RANK) ligand (RANKL). Anti-RANKL treatment for bone metastasis has been widely accepted for solid tumors. However, the mechanism of OPG-RANKL-RANK signaling in systemic colorectal cancer (CRC) metastasis remains unclear. In this study, we investigated the relevance and function of OPG expression in CRC liver metastasis. First, we performed in silico analysis using The Cancer Genome Atlas public database and found that lower OPG expression in CRC was associated with poor overall survival. Immunohistochemistry analyses using resected specimen from patients with CRC in our institute confirmed the result. Patient-matched primary CRC and liver metastases showed a significant downregulation of OPG expression in metastatic lesions. In CRC cell lines, OPG expression did not suppress cell proliferation and migration. However, OPG expression inhibited macrophage migration by suppressing the RANKL-RANK pathway. Moreover, in vivo mouse liver metastasis models showed that OPG expression in CRC cells suppressed liver metastases. In addition, treatment with an anti-RANKL neutralizing antibody also suppressed liver metastases. These results showed that downregulation of OPG expression in CRC cells promotes liver metastasis by activating tumor-associated macrophage, which can become a candidate for targeted therapy with anti-RANKL neutralizing antibody for CRC liver metastasis.

Neutrophil Extracellular Traps Promote Metastases of Colorectal Cancers through Activation of ERK Signaling by Releasing Neutrophil Elastase.

Neutrophil extracellular traps (NETs) play important roles in host immunity, as there is increasing evidence of their contribution to the progression of several types of cancers even though their role in colorectal cancers (CRCs) remains unclear. To investigate the clinical relevance of NETs in CRCs, we examined the expression of citrullinated histone H3 using immunohistochemistry and preoperative serum myeloperoxidase-DNA complexes in CRC patients using an enzyme-linked immunosorbent assay. High expression of intratumoral or systemic NETs was found to correlate with poor relapse-free survival (RFS), for which it is an independent prognostic factor. In vitro investigations of CRC cells (HCT116, HT29) revealed that NETs did not affect their proliferation but did promote the migration of CRC cells mediated by neutrophil elastase (NE) released during NETosis to increase extracellular signal-regulated kinase (ERK) activity. In vivo experiments using nude mice (KSN/slc) revealed that NE inhibition suppressed liver metastases in CRC cells, although it did not affect the growth of subcutaneously implanted tumors. Taken together, these results suggest that NET formation correlates with poor prognoses of patients with CRC and that the inhibition of NE could be a potential therapy for CRC metastases.

A case of long-term survival treated with three metastasectomies and two subsequent adjuvant nivolumab therapies for recurrent malignant melanoma of the esophagus.

BACKGROUND: The treatment strategy for metastatic lesions of primary malignant melanoma of the esophagus (PMME) is currently determined on a case-by-case basis, based on the National Comprehensive Cancer Network (NCCN) guidelines for cutaneous melanoma. The NCCN guidelines state that resection should be considered in patients with resectable metastatic recurrence. Herein, we report a case of long-term survival treated with three metastasectomies and two subsequent adjuvant nivolumab therapies for the metastatic recurrence of PMME. CASE PRESENTATION: A 65-year-old female patient with PMME underwent thoracoscopic subtotal esophagectomy, gastric tube reconstruction via the posterior mediastinal route, and cervical esophagogastric anastomosis. Histopathological examination of the resected specimen revealed that the tumor was PMME with tumor invasion into the muscularis propria and no lymph node metastasis. At the age of 68 years, she developed intestinal invagination due to jejunal metastasis of malignant melanoma and underwent resection of the jejunum. Histopathological examination of the resected specimen revealed two metastases of malignant melanoma in the jejunum and one metastasis to the mesenteric lymph node. At the age of 75 years, a recurrence of malignant melanoma was found in the cervical esophagus. She underwent thoracoscopic mobilization of the gastric tube and esophagus followed by cervical esophagectomy and reconstruction with a free jejunum flap. She received 24 courses of nivolumab therapy for 1 year as a postoperative adjuvant therapy. Subsequently, at the age of 78 years, an enlarged left cervical lymph node and a mass in the right lower lobe of the lung were found. She underwent left cervical lymph node dissection and thoracoscopic wedge resection of the right lung. Histopathological examination of the resected specimens revealed that both tumors were metastases of malignant melanoma. At age 79 years, she received eight courses of nivolumab therapy as a second postoperative adjuvant therapy, with no sign of recurrence in a 9-month follow-up period after the third metastasectomy. CONCLUSION: In cases of metastatic recurrence of PMME, aggressive resection of oligometastasis with postoperative adjuvant nivolumab therapy may result in long-term survival.

Targeting Asparagine Synthetase in Tumorgenicity Using Patient-Derived Tumor-Initiating Cells.

Reprogramming of energy metabolism is regarded as one of the hallmarks of cancer; in particular, oncogenic RAS has been shown to be a critical regulator of cancer metabolism. Recently, asparagine metabolism has been heavily investigated as a novel target for cancer treatment. For example, Knott et al. showed that asparagine bioavailability governs metastasis in a breast cancer model. Gwinn et al. reported the therapeutic vulnerability of asparagine biosynthesis in KRAS-driven non-small cell lung cancer. We previously reported that KRAS-mutated CRC cells can adapt to glutamine depletion through upregulation of asparagine synthetase (ASNS), an enzyme that synthesizes asparagine from aspartate. In our previous study, we assessed the efficacy of asparagine depletion using human cancer cell lines. In the present study, we evaluated the clinical relevance of asparagine depletion using a novel patient-derived spheroid xenograft (PDSX) mouse model. First, we examined ASNS expression in 38 spheroid lines and found that 12 lines (12/37, 32.4%) displayed high ASNS expression, whereas 26 lines (25/37, 67.6%) showed no ASNS expression. Next, to determine the role of asparagine metabolism in tumor growth, we established ASNS-knockdown spheroid lines using lentiviral short hairpin RNA constructs targeting ASNS. An in vitro cell proliferation assay demonstrated a significant decrease in cell proliferation upon asparagine depletion in the ASNS-knockdown spheroid lines, and this was not observed in the control spheroids lines. In addition, we examined asparagine inhibition with the anti-leukemia drug L-asparaginase (L-Asp) and observed a considerable reduction in cell proliferation at a low concentration (0.1 U/mL) in the ASNS-knockdown spheroid lines, whereas it exhibited limited inhibition of control spheroid lines at the same concentration. Finally, we used the PDSX model to assess the effects of asparagine depletion on tumor growth in vivo. The nude mice injected with ASNS-knockdown or control spheroid lines were administered with L-Asp once a day for 28 days. Surprisingly, in mice injected with ASNS-knockdown spheroids, the administration of L-Asp dramatically inhibited tumor engraftment. On the other hands, in mice injected with control spheroids, the administration of L-Asp had no effect on tumor growth inhibition at all. These results suggest that ASNS inhibition could be critical in targeting asparagine metabolism in cancers.

Quantitative Analysis of Colonic Perfusion Using ICG Fluorescence Angiography and Its Consequences for Anastomotic Healing in a Rat Model.

Forty-three rats were randomly assigned to the following four groups: non-ischemic group (Control Group), 1 cm-long ischemic group (Group 1), 2 cm-long ischemic group (Group 2), and 3 cm-long ischemic group (Group 3). The rates of AL were 0% (0/10) in the Control Group, 22.2% (2/9) in Group 1, 25% (2/8) in Group 2, and 50% (4/8) in Group 3. The bursting pressure of the Control Group was significantly higher than that of the other groups (p < 0.01). Regarding the pathological findings, the granulation thickness and the number of blood vessels at the anastomosed site were significantly higher in the Control Group than in Group 3 (p < 0.05). Receiver operating characteristics analysis revealed that Slope was the most significant predictor of AL, with an area under the curve of 0.861. When the cutoff value of Slope was 0.4, the sensitivity and specificity for the prediction of AL were 75% and 81.4%, respectively. Quantitative analysis of ICG fluorescence angiography could predict AL in a rat model.

McKeown esophagectomy with concomitant median arcuate ligament release in a case of esophageal cancer with celiac artery stenosis.

BACKGROUND: The celiac artery stenosis due to compression by median arcuate ligament (MAL) has been reported in many cases of pancreaticoduodenectomy, but not in cases of esophagectomy. Recently, the celiac artery stenosis due to MAL or arteriosclerosis has been reported to be associated with the gastric tube necrosis or anastomotic leakage following Ivor-Lewis esophagectomy. Herein, we present the first reported case of esophageal cancer with celiac artery stenosis due to compression by the MAL successfully treated by McKeown esophagectomy and gastric tube reconstruction following prophylactic MAL release. CASE PRESENTATION: A 72-year-old female patient was referred to our department for esophagectomy. The patient had received two courses of neoadjuvant chemotherapy with 5-FU and cisplatin for T2N0M0 squamous cell carcinoma of the middle esophagus. Preoperative contrast-enhanced computed tomography (CECT) showed celiac artery stenosis due to compression by the MAL. The development of collateral arteries around the pancreatic head was observed without evidence of aneurysm formation. The patient reported no abdominal symptoms. After robot-assisted esophagectomy with mediastinal lymphadenectomy, gastric mobilization, supra-pancreatic lymphadenectomy, and preparation of the gastric tube were performed under laparotomy. Subsequently, the MAL was cut, and released to expose the celiac artery. Improved celiac artery blood flow was confirmed by decreased pulsatility index on intraoperative Doppler sonography. The operation was completed with the cervical esophagogastric anastomosis following cervical lymphadenectomy. Postoperative CECT on postoperative day 7 demonstrated increased celiac artery patency. The patient had an uncomplicated postoperative course thereafter. CONCLUSIONS: Prophylactic MAL release may be considered in patients with celiac artery stenosis due to compression by the MAL on preoperative CECT for esophagectomy.

Dual blockade of macropinocytosis and asparagine bioavailability shows synergistic anti-tumor effects on KRAS-mutant colorectal cancer.

Mutations of KRAS gene are found in various types of cancer, including colorectal cancer (CRC). Despite intense efforts, no pharmacological approaches are expected to be effective against KRAS-mutant cancers. Macropinocytosis is an evolutionarily conserved actin-dependent endocytic process that internalizes extracellular fluids into large vesicles called macropinosomes. Recent studies have revealed macropinocytosis's important role in metabolic adaptation to nutrient stress in cancer cells harboring KRAS mutations. Here we showed that KRAS-mutant CRC cells enhanced macropinocytosis for tumor growth under nutrient-depleted conditions. We also demonstrated that activation of Rac1 and phosphoinositide 3-kinase were involved in macropinocytosis of KRAS-mutant CRC cells. Furthermore, we found that macropinocytosis was closely correlated with asparagine metabolism. In KRAS-mutant CRC cells engineered with knockdown of asparagine synthetase, macropinocytosis was accelerated under glutamine-depleted condition, and albumin addition could restore the glutamine depletion-induced growth suppression by recovering the intracellular asparagine level. Finally, we discovered that the combination of macropinocytosis inhibition and asparagine depletion dramatically suppressed the tumor growth of KRAS-mutant CRC cells in vivo. These results indicate that dual blockade of macropinocytosis and asparagine bioavailability could be a novel therapeutic strategy for KRAS-mutant cancers.

Effect of herbal medicine daikenchuto on gastrointestinal symptoms following laparoscopic colectomy in patients with colon cancer: A prospective randomized study.

We conducted a prospective randomized study to investigate the effect of daikenchuto (DKT) on abdominal symptoms following laparoscopic colectomy in patients with left-sided colon cancer. Patients who suffered from abdominal pain or distention on postoperative day 1 were randomized to either the DKT group or non-DKT group. The primary endpoints were the evaluation of abdominal pain, abdominal distention, and quality of life. The metabolome and gut microbiome analyses were conducted as secondary endpoints. A total of 17 patients were enrolled: 8 patients in the DKT group and 9 patients in the non-DKT group. There were no significant differences in the primary endpoints and postoperative adverse events between the two groups. The metabolome and gut microbiome analyses showed that the levels of plasma lipid mediators associated with the arachidonic acid cascade were lower in the DKT group than in the non-DKT group, and that the relative abundance of genera Serratia and Bilophila were lower in the DKT group than in the non-DKT group. DKT administration did not improve the abdominal symptoms following laparoscopic colectomy. The effects of DKT on metabolites and gut microbiome have to be further investigated.

Disruption of CCR1-mediated myeloid cell accumulation suppresses colorectal cancer progression in mice.

Tumor-stromal interaction is implicated in tumor progression. Although CCR1 expression in myeloid cells could be associated with pro-tumor activity, it remains elusive whether disruption of CCR1-mediated myeloid cell accumulation can suppress tumor progression. Here, we investigated the role of CCR1 depletion in myeloid cells in two syngeneic colorectal cancer mouse models: MC38, a transplanted tumor model and CMT93, a liver metastasis model. Both cells induced tumor accumulation of CCR1+ myeloid cells that express MMP2, MMP9, iNOS, and VEGF. Lack of the Ccr1 gene in host mice dramatically reduced MC38 tumor growth as well as CMT93 liver metastasis. To delineate the contribution of CCR1+ myeloid cells, we performed bone marrow (BM) transfer experiments in which sub-lethally irradiated wild-type mice were reconstituted with BM from either wild-type or Ccr1-/- mice. Mice reconstituted with Ccr1-/- BM exhibited marked suppression of MC38 tumor growth and CMT93 liver metastasis, compared with control mice. Consistent with these results, administration of a neutralizing anti-CCR1 monoclonal antibody, KM5908, significantly suppressed MC38 tumor growth and CMT93 liver metastases. Our findings highlight the importance of the application of CCR1 blockade as a therapeutic strategy.

Loss of SMAD4 Promotes Colorectal Cancer Progression by Recruiting Tumor-Associated Neutrophils via the CXCL1/8-CXCR2 Axis.

PURPOSE: SMAD4 is a key transcriptional factor of TGFß signaling and acts as a tumor suppressor in colorectal cancer. In the present study, we explored the immunologic effect of SMAD4 on the tumor microenvironment. EXPERIMENTAL DESIGN: Using 99 clinical specimens and human colorectal cancer cell lines, we investigate the relationship between SMAD4 expression and neutrophil accumulation. We immunohistochemically analyzed expression of SMAD4, CXCL1, CXCL8, CXCR2, and other proteins with clinical specimens. Finally, we determined the serum levels of CXCL1 and CXCL8 in 125 patients with colorectal cancer. RESULTS: SMAD4 knockdown from human colorectal cancer cells upregulated the expression of CXCL1 and CXCL8, which recruited neutrophils to colorectal cancer tumor via CXCR2. In turn, when neutrophils were exposed to the supernatant of SMAD4-negative colorectal cancer cells, they produced a large amount of CXCL1 and CXCL8 by themselves in vitro. In human clinical specimens, we found that neutrophil infiltration into the peritumoral stroma was more marked in SMAD4-negative colorectal cancer compared with that in SMAD4-positive colorectal cancer, and that both CXCL1 and CXCL8 were abundantly expressed in the tumor-infiltrating neutrophils. Neutrophils isolated from primary colorectal cancer expressed significantly higher levels of CXCL1 and CXCL8 than did those isolated from peripheral blood. Furthermore, tumor-infiltrating neutrophils expressed MMP2 and MMP9 in addition to ARG1 and IDO. Serum CXCL8 level was significantly higher in colorectal cancer patients, especially those at stage II/III, and statistical analysis indicated a high CXCL8 level was associated with a shorter overall survival and relapse-free survival. CONCLUSIONS: Blockade of the CXCL1/8-CXCR2 axis could be a novel therapeutic approach against SMAD4-negative colorectal cancer.

Chronic inguinal pain after laparoscopic intraperitoneal onlay mesh (IPOM) repair for inguinal hernia treated successfully with laparoscopic selective neurectomy: A case report.

INTRODUCTION: Laparoscopic intraperitoneal onlay mesh (IPOM) repair is occasionally used for inguinal hernia repair. Here, we report a case of chronic neuropathic pain after laparoscopic IPOM repair for inguinal hernia, which was treated successfully with laparoscopic selective neurectomy. PRESENTATION OF CASE: A 59-year-old man with bilateral inguinal hernia underwent laparoscopic repair. Transabdominal preperitoneal repair was performed on the left side, whereas IPOM repair was performed on the right side due to a peritoneal defect. At postoperative month 1, he presented with severe pain and numbness distributed from the right inguinal region to the inner thigh region. The symptoms had persisted for 1year despite medical treatment. We diagnosed that the symptoms might be due to the entrapment of nerves in the contracted mesh, and performed a second surgery via laparoscopic approach 13 months after the first surgery. On laparoscopic exploration, the lateral side of the mesh was contracted and involved nerve branches. We ligated and cut off these nerve branches. His symptoms resolved immediately after the surgery. At postoperative month 12, he has passed without any pain, numbness, and hernia recurrence. DISCUSSION: Laparoscopic exploration would be useful to figure out chronic neuropathic pain after laparoscopic inguinal hernia repair. CONCLUSION: Laparoscopic IPOM repair for inguinal hernia should be avoided as much as possible because it may cause chronic neuropathic pain. Laparoscopic selective neurectomy is an option for patients with chronic neuropathic pain after laparoscopic hernia repair.

Successful treatment for patients with chronic orchialgia following inguinal hernia repair by means of meshoma removal, orchiectomy and triple-neurectomy.

INTRODUCTION: Orchialgia following inguinal hernia repair is rare complication and still challenging since there has been no established surgical treatment because of complexity of nerve innervation to the testicular area. Herein we report a case of postoperative orchialgia following Lichtenstein repair, which was successfully treated by mesh removal, orchiectomy and triple neurectomy. CASE PRESENTATION: A 65-year-old man was referred to our department because of chronic right orchialgia following Lichtenstein hernia repair. He walked with a limp and was unable to walk a long distance. Physical examination revealed the presence of meshoma in the groin area and hypoesthesia in the anterior skin of the right scrotum. His right testis was completely atrophic and located not in the scrotum but in the subcutaneous regions of right groin. He was diagnosed as both neuropathic and nociceptive orchialgia and underwent meshoma removal, triple-neurectomy, and orchiectomy to address these issues. Pathological examination revealed that meshoma was integrated with the structures of the spermatic cord, leading to foreign-body reaction and fibrosis around the genital branch of genitofemoral nerve. The resected right testis was completely-scarred without ischemic changes. Orchialgia disappeared immediately after operation and he was able to walk without a limp. DISCUSSIONS: It is important to distinguish between nociceptive and neuropathic orchialgia. Neuroanatomic understanding is essential to guide treatment options. Orchiectomy is an option but should be reserved for refractory cases with evidence of nociceptive pain accompanied by anatomical changes. CONCLUSIONS: Triple neurectomy should be considered in patients with neuropathic orchialgia.

[Oral capecitabine as postoperative adjuvant chemotherapy in stage III colon cancer patients].

Capecitabine(Xeloda®)has been a global standard drug for the treatment of colon cancer since large randomized controlled trials demonstrated its efficacy and safety in treating patients suffering from the disease. Few studies have been conducted to assess the effects of oral capecitabine treatment on Japanese patients. Therefore, we conducted this study to evaluate oral capecitabine as postoperative adjuvant chemotherapy in 50 patients who underwent surgery for stage III colon cancer at our department. Patients received an 8 courses treatment with capecitabine during the study, and the incidence of adverse events, treatment completion rate, and treatment compliance were assessed. Adverse events were reported in a total of 46 patients(92%). The most common adverse event was hand foot syndrome(HFS), reported in 39 patients(78%), whereas bone-marrow toxicity and diarrhea were reported in as few as 2(4%)and 3(6%)patients, respectively. Both these events were mild in severity, and no patients required hospitalization, nor were they associated with treatment-related deaths. The median treatment duration was 8 courses ranging from 3 to 8 courses, and the 8 courses treatment completion rate was 96%. The relative dose intensity, which was used as a treatment compliance index, is expressed as the actual dose taken by the patient divided by the dose planned at baseline. The median and mean of the relative dose intensity were 100%(ranging from 37% to 100%)and 93%, respectively. The results of this study showed that the safety profile of oral capecitabine therapy was generally favorable, with a lower incidence and lesser severity of life-threatening bone-marrow toxicity and diarrhea, although the treatment is still associated with frequent HFS. This is the great advantage of capecitabine when it is used as postoperative adjuvant chemotherapy for gastrointestinal cancer. Indeed, a satisfactory treatment completion rate was achieved in this study while maintaining a sufficient dose and treating HFS, by reducing the dose, interrupting treatment, or providing appropriate corrective measures.