Bimodal role of Kupffer cells during colorectal cancer liver metastasis

Kupffer cells (KCs) are resident liver macrophages that play a crucial role in liver homeostasis and in the pathogenesis of liver disease. Evidence suggests KCs have both stimulatory and inhibitory functions during tumor development but the extent of these functions remains to be defined. Using KC depletion studies in an orthotopic murine model of colorectal cancer (CRC) liver metastases we demonstrated the bimodal role of KCs in determining tumor growth. KC depletion with gadolinium chloride before tumor induction was associated with an increased tumor burden during the exponential growth phase. In contrast, KC depletion at the late stage of tumor growth (day 18) decreased liver tumor load compared with non-depleted animals. This suggests KCs exhibit an early inhibitory and a later stimulatory effect. These two opposing functions were associated with changes in iNOS and VEGF expression as well as T-cell infiltration. KC depletion at day 18 increased numbers of CD3⁺ T cells and iNOS-expressing infiltrating cells in the tumor, but decreased the number of VEGF-expressing infiltrating cells. These alterations may be responsible for the observed reduction in tumor burden following depletion of pro-tumor KCs at the late stage of metastatic growth. Taken together, our results indicate that the bimodal role of KC activity in liver tumors may provide the key to timing immunomodulatory intervention for the treatment of CRC liver metastases.     

Kupffer cells and liver metastasis

Kupffer cells, tissue-fixed macrophages located in the sinusoids of the liver, represent the highest concentration of mononuclear phagocytes in the body. Their ability to act as scavengers of particulate material in the blood has given rise to speculation that they play a role in controlling hepatic metastases derived from blood-borne tumor cells. Circumstantial evidence for such a role has been obtained from animal studies where Kupffer cell function has been compromised or inhibited, and from anecdotal clinical observations. Current evidence suggests that Kupffer cells are capable of nonspecifically climinating some circulating tumor cells from the circulation via phagocytosis. This surveillance mechanism would appear to be limited in capacity, and subject to a number of external factors. Recent studies have demonstrated that Kupffer cells can be activated to a tumoricidal state via the administration of biological response modifiers such as gamma interferon or muramyl peptides. The localization of liposomes within Kupffer cell after systemic administration has provided a considerable stimulus for the efficient targeting of macrophage-activating compounds to these cells. Such therapeutic intervention, while capable of inducing Kupffer cell tumoricidal activity in situ and inhibiting tumor growth, is limited with respect to the location of the tumor cells (sinusoidal versus parenchymal) and to the size of the metastatic nodule. Therapeutic intervention using liposomes containing macrophage-activating agents may only be of benefit in patients with minimal tumor load who are at risk for hepatic metastases, rather than those patients who already have clinically detectable liver tumors.     

The renin angiotensin system regulates Kupffer cells in colorectal liver metastases

Blockade of the renin angiotensin system (RAS) can inhibit tumor growth and this may be mediated via undefined immunomodulatory actions. This study investigated the effects of RAS blockade on liver macrophages (Kupffer cells; KCs) in an orthotopic murine model of colorectal cancer (CRC) liver metastases. Here we showed that pharmacological targeting of the RAS [ANG II (31.25 µg/kg/h i.p.), ANG-(1–7) (24 µg/kg/h i.p.) or the ACE inhibitor; captopril (750 mg/kg/d i.p.)] altered endogenous KC numbers in the tumor-bearing liver throughout metastatic growth. Captopril, and to a lesser extent ANG-(1–7), increased KC numbers in the liver but not tumor. KCs were found to express the key RAS components: ACE and AT1R. Treatment with captopril and ANG II increased the number of AT1R-expressing KCs, although total KC numbers were not affected by ANG II. Captopril (0.1 µM) also increased macrophage invasion in vitro. Additionally, captopril was administered with KC depletion before tumor induction (day 0) or at established metastatic growth (day 18) using gadolinium chloride (GdCl_3; 20 mg/kg). Livers were collected at day 21 and quantitative stereology used as a measure of tumor burden. Captopril reduced growth of CRC liver metastases. However, when captopril was combined with early KC depletion (day 0) tumor growth was significantly increased compared with captopril alone. In contrast, late KC depletion (day 18) failed to influence the anti-tumor effects of captopril. The result of these studies suggests that manipulation of the RAS can alter KC numbers and may subsequently influence progression of CRC liver metastases.     

Interactions between rat colon carcinoma cells and Kupffer cells during the onset of hepatic metastasis

Liver sinusoids harbor populations of 2 important types of immunocompetent cells, Kupffer cells (KCs) and natural killer (NK) cells, which are thought to play an important role in controlling hepatic metastasis in the first 24 hr upon arrival of the tumor cells in the liver. We studied the early interaction of KCs, NK and CC531s colon carcinoma cells in a syngeneic rat model by confocal laser scanning microscopy. Results showed a minority of KCs (19% periportal and 7% pericentral) involved in the interaction with 94% of tumor cells and effecting the phagocytosis of 92% of them. NK cell depletion decreased the phagocytosis of tumor cells by KCs by 33% over a period of 24 hr, leaving 35% of the cancer cells free, as compared to 6% in NK-positive rats. Surviving cancer cells were primarily located close to the Glisson capsule, suggesting that metastasis would initiate from this region.     

Angiotensin II subtype 1a receptor signaling in resident hepatic macrophages induces liver metastasis formation

Liver metastases from colorectal cancer (CRC) are a clinically significant problem. The renin–angiotensin system is involved in tumor growth and metastases. This study was designed to evaluate the role of angiotensin II subtype receptor 1a (AT1a) in the formation of liver metastasis in CRC. A model of liver metastasis was developed by intrasplenic injection of mouse colon cancer (CMT‐93) into AT1a knockout mice (AT1aKO) and wild‐type (C57BL/6) mice (WT). Compared with WT mice, the liver weight and liver metastatic rate were significantly lower in AT1aKO. The mRNA levels of CD31, transforming growth factor‐ β1 (TGF‐β1), and F4/80 were suppressed in AT1aKO compared with WT. Double immunofluorescence analysis showed that the number of accumulated F4/80⁺ cells expressing TGF‐β1 in metastatic areas was higher in WT than in AT1aKO. The AT1aKO bone marrow (BM) (AT1aKO‐BM)→WT showed suppressed formation of liver metastasis compared with WT‐BM→WT. However, the formation of metastasis was further suppressed in WT‐BM→AT1aKO compared with AT1aKO‐BM→WT. In addition, accumulated F4/80⁺ cells in the liver metastasis were not BM‐derived F4/80⁺ cells, but mainly resident hepatic F4/80⁺ cells, and these resident hepatic F4/80⁺ cells were positive for TGF‐β1. Angiotensin II enhanced TGF‐β1 expression in Kupffer cells. Treatment of WT with clodronate liposomes suppressed liver metastasis by diminishing TGF‐β1⁺F4/80⁺ cells accumulation. The formation of liver metastasis correlated with collagen deposition in the metastatic area, which was dependent on AT1a signaling. These results suggested that resident hepatic macrophages induced liver metastasis formation by induction of TGF‐β1 through AT1a signaling.     

Historical perspective: Two decades of progress in treating metastatic colorectal cancer

Colorectal cancer is the third most commonly diagnosed cancer in the United States. While screening methods strive to improve rates of early stage detection, 25% of patients have metastatic disease at the time of diagnosis, with the most common sites being the liver, lung, and peritoneum. While once perceived as hopeless, the last two decades have seen substantial strides in the medical, surgical, and regional therapies to treat metastatic disease offering significant improvements in survival.     

DC-CIK对结直肠癌根治术后肝转移患者疗效和循环肿瘤细胞的影响

目的: 探讨树突状细胞-细胞因子诱导的杀伤细胞（dendritic cell-cytokine induced killer cell, DC-CIK）对结直肠癌（colorectal carcinoma, CRC）根治术后肝转移患者循环肿瘤细胞（circulating tumor cell, CTC）数量、疗效和预后的影响。方法: 回顾性分析2009 年7 月至2015 年12 月在解放军第309 医院普通外科采用DC-CIK+常规疗法治疗的CRC根治术后肝转移患者62例（DC-CIK组）和同期未接受DC-CIK治疗的70 例患者（常规组）的临床资料,同时抽取部分患者（DC-CIK组21 例,常规组24 例）的外周静脉血,用CellSearch R 免疫磁珠技术检测血中CTC的数量,分析比较两组患者的疗效和预后。结果：DC-CIK 组治疗后患者CTC数量明显低于治疗前[ （1.0±1.1）vs（2.7±2.0）个,P<0.01],而常规组CTC变化不明显（P>0.05）;DC-CIK组肝转移灶手术切除率、治疗客观有效率、患者无进展生存和总生存均显著优于常规组（P<0.05 或P<0.01）。结论：DC-CIK可减少CRC根治术后肝转移患者CTC数量并延长患者生存时间,在规范化实施的前提下具有可靠的抗肿瘤效果。     

Transient Kupffer cell depletion and subsequent replacement by infiltrating monocyte-derived cells does not alter the induction or progression of hepatocellular carcinoma

Due to a combination of rapid disease progression and the lack of curative treatment options, hepatocellular carcinoma (HCC) is one of the deadliest cancers worldwide. Infiltrated, monocyte-derived, tumor-associated macrophages are known to play a role in HCC pathogenesis, but the involvement of Kupffer cells (KCs) remains elusive. Here, we used the Clec4F-diphteria toxin receptor transgenic mouse model to specifically investigate the effect of KC depletion on HCC initiation, progression and neoplastic growth following liver resection. For this purpose, several HCC mouse models with varying underlying etiologies were used and partial hepatectomy was performed. Our results show that in HCC, developed on a fibrotic or non-alcoholic steatohepatitis background, depletion of embryonic KCs at the onset of HCC induction and the subsequent replacement by monocyte-derived KCs does not affect the tumor burden, tumor microenvironment or the phenotype of isolated KCs at end-stage disease. In non-chronic liver disease-associated diethylnitrosamine-induced HCC, ablation of Clec4F⁺ KCs did not alter tumor progression or neoplastic growth following liver resection. Our results show that temporal ablation of resident KCs does not impact HCC pathogenesis, neither in the induction phase nor in advanced disease, and indicate that bone marrow-derived KCs are able to swiftly repopulate the available KC niche and adopt their phenotype.     

树突状细胞疫苗联合射频消融术治疗结直肠癌肝转移临床疗效

目的:探讨树突状细胞(dendritic cell,DC)疫苗联合射频消融术(radiofrequency ablation,RFA)治疗结直肠癌肝转移(colorectal liver metastases,CRLM)患者的临床疗效.方法:选取2012年8月至2014年8月在解放军第八一医院确诊并行RFA治疗的46例CRLM患者,其中26例进行DC疫苗联合RFA治疗(DC-RFA组),20例单纯进行RFA治疗(RFA组),比较两组患者近期、远期疗效,免疫功能,安全性及生活质量改善情况.结果:(1)DC-RFA组总有效率明显高于对照组(92.31％ vs 70.00％,P＜0.05);DC-RFA组与RFA组治疗后6个月生存率分别为96.15％、90.00％;1、2年生存率DC-RFA组略优于RFA组(P＞0.05);(2)DC-RFA组外周血CD3+、CD4+、CD4+/CD8+百分比明显升高(P＜0.05),CD8+数值降低(P＞0.05);RFA组治疗后外周血CD3+、CD4+及CD4 +/CD8+百分比明显升高(P＜0.05),CD8+数值升高(P＞0.05);(3)DC-RFA组治疗后仅有2例低热,1例过敏反应,对症处理后均恢复正常;(4)DC-RFA组患者生活质量有所提高,尤其是疼痛控制和精神状态方面.结论:DC疫苗联合RFA治疗CRLM患者可提高单纯RFA治疗的效果、延长生存期、提高机体免疫功能,同时可以有效改善患者生活质量,且治疗安全可靠.     

Engulfment and cell motility protein 1 fosters reprogramming of tumor-associated macrophages in colorectal cancer

Functional reprogramming of tumor-associated macrophages (TAMs) is crucial to their potent tumor-supportive capacity. However, the molecular mechanism behind the reprogramming process remains poorly understood. Here, we identify engulfment and cell motility protein 1 (ELMO1) as a crucial player for TAM reprogramming in colorectal cancer (CRC). The expression of ELMO1 in stromal but not epithelial tumor cells was positively associated with advanced clinical stage and poor disease-free survival in CRC. An increase in ELMO1 expression was specifically found in TAMs, but not in other multiple nonmalignant stromal cells. Gain- and loss-of-function assays indicated ELMO1 reprogrammed macrophages to a TAM-like phenotype through Rac1 activation. In turn, ELMO1-reprogrammed macrophages were shown to not only facilitate the malignant behaviors of CRC cells but exhibited potent phagocytosis of tumor cells. Taken together, our work underscores the importance of ELMO1 in determining functional reprogramming of TAMs and could provide new insights on potential therapeutic strategies against CRC.     

Vascular density in colorectal liver metastases increases after removal of the primary tumor in human cancer patients

In animal models, explosive growth of metastases after removal of the primary tumor has been attributed to abolishment of angiogenesis inhibition. We investigated the influence of (removal of) the primary tumor on vascularization of liver metastases in human colorectal cancer patients. We analyzed vascular density in synchronous liver metastases from patients with the primary tumor in situ, in synchronous metastases from patients with the primary tumor resected and in metachronous metastases. In a limited number of cases, biopsies from metastases from the same patient before and within 3 months after resection were analyzed. In addition, vascular density in metastases was compared to the vascular density in the corresponding primary tumor. Peritumoral and intratumoral vascular density were determined by staining for endothelial antigens CD31 and CD34, respectively. Both peritumoral and intratumoral vascular density were elevated in synchronous metastases from patients with the primary tumor removed compared to synchronous metastases from patients with the primary tumor in situ. Comparable results were observed in patients with metachronous metastases. An increase in vascular density after resection of the colorectal malignancy was also observed in biopsies taken from the same patient before and after tumor resection. Remarkably, vascular density in the liver metastases was always lower than that in the corresponding primary tumor. Our data show for the first time in humans that the presence of a primary tumor is correlated with decreased vascularization of its distant metastases. Resection of the primary tumor results in an increased vascularization of metastatic lesions.     

Adjuvant, adoptive immunotherapy with tumor infiltrating lymphocytes plus interleukin-2 after radical hepatic resection for colorectal liver metastases: 5-year analysis

BACKGROUND AND OBJECTIVES: Conventional chemotherapy has not proven effective in improving long-term results of surgery for liver metastases from colorectal cancer. We assessed the usefulness of immunotherapy with tumor infiltrating lymphocytes (TIL) plus Interleukin-2 (IL-2) as adjuvant treatment. METHODS: Between 1995 and 1998, 47 patients were enrolled onto a prospective protocol; 25 entered the treatment group (A) and 22 entered the control group (B). All patients had undergone radical liver resection. TIL obtained from surgical specimens from group A patients were cultured and activated in vitro with IL-2, then reinfused into the patients with IL-2. We investigated pre- and post-IL-2 stimulation expression of T cell receptor (TCR) zeta- and epsilon-chains, p56(lck), Fas, and Fas-L by TIL immunostaining. RESULTS: Fourteen patients from group A (56%) received immunotherapy; 14 from group B (60%) underwent conventional chemotherapy, and the remaining 19 patients did not receive any treatment. No significant differences between the two groups were found in the actuarial and disease-free survival (DSF) rates after 1, 3, and 5 years. After IL-2 exposure, TCR zeta-chain expression significantly increased (P = 0.001); An increase in TCR epsilon-chain expression (P = 0.04), and p56(lck) (P = 0.03) was detected; TCR epsilon-chain expression was significantly increased in disease-free patients compared to those who relapsed (P = 0.04). Fas-L expression was correlated with the TCR epsilon-chain and p56(lck) levels (P = 0.05). CONCLUSIONS: Our data suggest that we are still a long way from being able to propose TIL + IL-2 treatment as an effective adjuvant therapy. However, the results confirm that the biological indicators examined could play an important role in modulating immunitary response against tumor cells.     

FOLFOX6 and bevacizumab in non-optimally resectable liver metastases from colorectal cancer

Background:

In patients with colorectal liver metastases (CLM) R0 resection significantly improves overall survival (OS).

Methods:

In this report, we present the results of a phase II trial of FOLFOX6+bevacizumab in patients with non-optimally resectable CLM. Patients received six cycles of FOLFOX6+ five of bevacizumab. Patients not achieving resectability received six additional cycles of each. A PET-CT was performed at baseline and again within 1 month after initiating treatment.

Results:

From September 2005 to July 2009, 21 patients were enrolled (Male/Female: 15/6; median age: 65 years). An objective response (OR) was documented in 12 cases (57.1% complete responses (CRs): 3, partial response (PR): 9); one patient died from toxicity before surgery. Thirteen patients underwent radical surgery (61.9%). Three (23%) had a pathological CR (pCR). Six patients (46.1%) experienced minor postsurgical complications. After a median 38.8-month follow-up, the median OS was 22.5 months. Patients achieving at least 1 unit reduction in Standard uptake value (SUV)max on PET-CT had longer progression-free survival (PFS) (median PFS: 22 vs 14 months, P=0.001).

Conclusions:

FOLFOX6+bevacizumab does not increase postsurgical complications, yields high rates of resectability and pCR. Early changes in PET-CT seem to be predictive of longer PFS.     

Morphologic analysis of microvessels in colorectal tumors with respect to the formation of liver metastases.

BACKGROUND AND OBJECTIVES: Overexpression of VEGF and proliferation of microvessels are strongly related to liver metastases, however, morphologic analyses of microvessels in liver metastases have not been reported. The purpose of the present study was to examine the correlation between liver metastases and the diameters of microvessel lumens in the tumor tissue. METHODS: Fifty-nine patients with liver metastases from colorectal cancers and 112 patients who underwent curative colorectal resection and survived without any recurrence were reviewed. Microvessel density (MVD) and the diameters of the lumens of individual microvessels were assessed. RESULTS: There was a significant difference in terms of the mean MVD of primary tumors between patients with liver metastases and those without liver metastases. The numbers of patients with liver metastases who had microvessels 100-200 microm in diameter and microvessels more than 200 microm in diameter were significantly greater than patients without liver metastases. Microvessels with lumens more than 100 microm in diameter were not detected in the liver metastatic lesion. CONCLUSION: Large microvessels in the primary tumor favor intravasation of cancer cells.     

Liver metastases from colorectal cancer: Technique of liver resection

Liver resection has become standard for the treatment of metastatic colorectal cancer (CRC): anterior approach, hanging manoeuvre, or total vascular exclusion techniques as well as 3‐dimensional imaging enable safe resections even in difficult cases. Furthermore, modern chemotherapy, portal vein embolization/ligation, and two‐stage procedures increase the resectability of metastasis, and repeat resections are feasible for recurrence. In addition to characteristics of the primary, CEA, extent of metastasis, resection margins, and extrahepatic disease, hilar lymph node metastases appear prognostic. J. Surg. Oncol. 2013;107:579–584. © 2012 Wiley Periodicals, Inc.     

结直肠癌循环肿瘤细胞的临床研究进展

结直肠癌是最常见的恶性肿瘤之一,大量患者死于术后转移和复发.循环肿瘤细胞是存在于患者外周血循环中的肿瘤细胞,是肿瘤发生远处转移的关键,与患者预后密切相关.本文综述近年来有关结直肠癌循环肿瘤细胞的临床研究进展.     

Effective delivery of chemotherapeutic nanoparticles by depleting host Kupffer cells

Although chemotherapeutic nanoparticles would confer various advantages, the majority of administrated nanoparticles are known to be spoiled by the reticuloendothelial system (RES). Intending to more effectively deliver therapeutic nanoparticles to target regions in vivo, host RES, especially Kupffer cells in the liver, have been depleted ahead of drug administration. To demonstrate this hypothesis, clodronate liposomes were preinjected into BALB/c nude mice for depletion of Kupffer cells 2 days before, and pegylated liposomal doxorubicin (Doxil) at the doses of 1.25, 2.5 and 5.0 mg/kg was administered. As a result, doxorubicin accumulation in the liver was decreased from 36 to 26% injected dose/organ by the Kupffer cells depletion, and consequently, the plasma concentration of doxorubicin was significantly enhanced threefold (from 11 to 33 μg/mL) on day 1 at 1.25 mg/kg-dose group. Doxorubicin accumulation in the tumor was increased from 0.78 to 3.0 μg/g-tissue on day 3, and tumor growth inhibition by Doxil was significantly boosted (tumor volumes from 751 to 482 mm(3) on day 24) by the Kupffer cells depletion. In conclusion, Kupffer cells depletion by clodronate liposomes enhanced the plasma concentration and antitumor effects of Doxil, and would be widely applicable for various clinical cancer chemotherapies using nanoparticles.     

Kupffer cells of cirrhotic rat livers sensitize colon cancer cells to Fas-mediated apoptosis

Metastasis of colorectal carcinomas rarely occurs in cirrhotic livers. Our study investigated the influence of activated Kupffer cells from cirrhotic rat livers on hepatic colonization and FasR-mediated apoptosis of colon cancer cells. A rat colon cancer cell line, RCN-9, was used to inoculate rat livers. Treatment with conditioned media of Kupffer cells isolated from CCl(4)-induced cirrhotic rat livers (cirrhotic KCM) significantly reduced the incidence of hepatic colonization of RCN-9 cells. In vitro cytotoxicity of Kupffer cells and tumour infiltrating lymphocytes (TILs) on RCN-9 cells was evaluated using [(3)H]-release assay. RCN-9 cells were resistant to cytotoxicity mediated by cirrhotic Kupffer cells, but were sensitized to TIL-mediated killing after treatment with cirrhotic KCM. The specific killing induced by TILs was FasR-mediated, as it was inhibited by ZB4, an antagonistic anti-FasR antibody. In agreement, cirrhotic KCM increased recombinant Fas ligand-induced apoptosis of RCN-9 cells, and up-regulated FasR expression on RCN-9 cells as evaluated by RT-PCR and flow cytometry. These findings suggest that Kupffer cells in cirrhotic livers sensitize metastatic colon cancer cells to FasR-mediated apoptosis by up-regulating the receptors, which thus prepare them to be eliminated by infiltrating lymphocytes.     

Evaluation of a follow-up programme after curative resection for colorectal cancer

Frequent liver imaging can detect liver metastases from colorectal cancer at an asymptomatic stage. © 1999 Cancer Research Campaign