Liver Micrometastases Research Articles

Survival impact of [225Ac]Ac-DOTATOC alpha-therapy in a preclinical model of pancreatic neuroendocrine tumor liver micrometastases.

A mouse model of liver metastases of pancreatic NETs was developed by intraportal injection of AR42J cells and explored using [68Ga]Ga-DOTATOC and [18F]F-FDG PET/MRI. Biodistribution study and radiation dosimetry of [225Ac]Ac-DOTATOC were determined in subcutaneous tumor-bearing NMRI-nude mice. Efficacy and toxicity were determined by intravenous injection of increasing activities of [225Ac]Ac-DOTATOC 10days after intraportal graft. Liver tumors showed a high uptake of [68Ga]Ga-DOTATOC and no uptake of [18F]F-FDG confirming the well-differentiated phenotype. All groups treated with [225Ac]Ac-DOTATOC showed a significant increase in overall survival compared with DOTATOC-treated mice, especially those treated with the highest activities: 53days with 240kBq (p = 0.0001), and 58days with 2 × 120kBq (p < 0.0001) vs 28days with non-radiolabeled DOTATOC. On blood tests, a transient and moderate decreased in white blood cells count after treatment and no severe hepatic or renal toxicity were observed after treatment which was consistent with pathological and radiation dosimetry findings. [225Ac]Ac-DOTATOC exhibit a favorable efficacy and toxicity profile in a mouse model of liver micrometastatic pancreatic NET.

A head-to-head comparison of 18F-FDG PET/CT and 18F-FDG PET/MR in patients with nasopharyngeal carcinoma under different disease settings.

Positron emission tomography/computed tomography (PET/CT) and magnetic resonance imaging (MRI) are complementary in staging of nasopharyngeal carcinoma (NPC). The combination of MRI and functional imaging from PET in PET/MR is promising in NPC management. Diagnostic performance of PET/CT and PET/MR was compared in 46 patients with histologically confirmed NPC under different disease scenarios, including primary non-metastatic cases, primary metastatic cases, recurrence and/or metastasis after treatment, and post-treatment follow-up cases. Forty-six patients underwent both PET/CT and PET/MR in the same day. Primary tumor extension into risk-stratified anatomic structures, retropharyngeal and cervical lymph node metastasis, distant metastasis and post-treatment follow-up results, were compared. For high-risk structures, PET/MR detected two more sides of tensor/levator veli palatine muscle involvement, one more case of clivus involvement, and ruled out 12 false-positive sides of prevertebral muscle involvement by PET/CT. For medium-risk structures, PET/MR detected four more sides of medial pterygoid muscle involvement. For low-risk structures, abnormal signal on massa lateralis atlantis was detected by PET/MR. PET/MR detected 14 more positive retropharyngeal lymph nodes and more liver micrometastases than PET/CT. Overall, PET/MR changed two patients' T staging. Positron emission tomography/MR outperforms PET/CT in delineating muscle, skull-base bone, and nodal involvement, and identifying liver micrometastases, may serve as a single-step staging modality for NPC.

Abstract 1168: Anti-CSF1R antibody monotherapy inhibits minimal residual disease in a novel immunocompetent murine colorectal cancer metastasis model

Abstract Background: The high mortality rate of colorectal cancer (CRC) is primarily due to metastatic disease. Microscopic minimal residual disease (MRD) detected in patients by circulating tumor DNA (ctDNA) may remain radiographically undetectable and persist following therapeutic intervention, resulting in recurrence. Preclinical models are urgently needed to elucidate the mechanisms whereby MRD escapes anti-tumor immune surveillance. We have developed and characterized a preclinical liver MRD model that recapitulates immune response, with the goal to discover novel strategies to improve outcomes for patients with ctDNA+ MRD. Methods: We generated genetically engineered mouse colonic organoids CDX2 CRE; Rosa26 LSL-CAS9-GFP with APC and TP53 mutation and performed intrasplenic injection in syngeneic C57BL/6 mice to recapitulate microsatellite stable CRC metastasis to the liver. Mice were monitored for radiographically detectable disease using MRI. Tumors were resected at various timepoints, with comparison between metastases at various sizes of development (dichotomized as micro- and macro-metastases). We performed multiplex immunofluorescence staining, single cell RNA seq, and spatial transcriptomic analysis to delineate the immune compartments of micro- and macro-metastasis in untreated mice. After identifying colony stimulating factor -1 receptor (CSF1R) as a target, we performed a vehicle controlled anti-CSF1R monotherapy study for 4 weeks. Treatment began at either 7 days before metastasis is radiographically detectable to mimic treatment of MRD, or 21 days post tumor engraftment. Results: Immune exclusion was confirmed through intratumoral CD45+ leukocyte densities, with transition to an immune excluded phenotype occurring above 0.5mm in size (defined as macro-metastases). In our preclinical model, immunosuppressive populations of macrophages (IBA1+CD163+) dominated in the micro-metastases. This observation was confirmed in 13 patients with paired micro- and macro-metastases to the liver (p=0.0065). Strikingly, anti-CSF1R treatment in the murine model eradicated IBA1+CD163+ macrophages (p&amp;lt;0.0001) and induced complete remission of liver micro-metastasis (0/7 mice with residual disease), in contrast to limited efficacy for macro-metastases (5/5 mice with progressed radiographically disease). Additionally, a long-term study confirmed 100% survival (5/5 mice) and no evidence of disease via MRI at 6 months upon anti-CSF1R treatment compared to vehicle (0/5 mice). Conclusions: We established a reproducible preclinical model of MRD with similar immunopathological features of MRD in patients. Our data further identified infiltration of immunosuppressive macrophage populations as an early determinant of MRD progression and indicated CSF1R as a potential new clinical therapeutic target for ctDNA+-defined MRD patients. Citation Format: Alaa M. Mohamed, Kyung Serk Cho, Melinda Soeung, Amanda Anderson, Jumanah Alshenaifi, Ganiraju Manyam, Oscar Villareal, Jennifer Davis, Will Norton, Sisi Gao, Christopher Bristow, Federica Carbone, Stefania Napolitano, Oluwadara Coker, Isha Khanduri, Justin Huang, Dipen M. Maru, Mauro Di Pilato, Ryan Sun, Linghua Wang, David G. Menter, Giannicola Genovese, Giulio Draetta, Natalie Fowlkes, Scott Kopetz. Anti-CSF1R antibody monotherapy inhibits minimal residual disease in a novel immunocompetent murine colorectal cancer metastasis model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1168.

Dual antiplatelet therapy inhibits neutrophil extracellular traps to reduce liver micrometastases of intrahepatic cholangiocarcinoma

The involvement of neutrophil extracellular traps (NETs) in cancer metastasis is being clarified, but the relationship between intrahepatic cholangiocarcinoma (iCCA) and NETs remains unclear. The presence of NETs was verified by multiple fluorescence staining in clinically resected specimens of iCCA. Human neutrophils were co-cultured with iCCA cells to observe NET induction and changes in cellular characteristics. Binding of platelets to iCCA cells and its mechanism were also examined, and their effects on NETs were analyzed in vitro and in in vivo mouse models. NETs were present in the tumor periphery of resected iCCAs. NETs promoted the motility and migration ability of iCCA cells in vitro. Although iCCA cells alone had a weak NET-inducing ability, the binding of platelets to iCCA cells via P-selectin promoted NET induction. Based on these results, antiplatelet drugs were applied to these cocultures in vitro and inhibited the binding of platelets to iCCA cells and the induction of NETs. Fluorescently labeled iCCA cells were injected into the spleen of mice, resulting in the formation of liver micrometastases coexisting with platelets and NETs. These mice were treated with dual antiplatelet therapy (DAPT) consisting of aspirin and ticagrelor, which dramatically reduced micrometastases. These results suggest that potent antiplatelet therapy prevents micrometastases of iCCA cells by inhibiting platelet activation and NET production, and it may contribute to a novel therapeutic strategy.

Abstract 3126: The secretome of colorectal cancer cell lines cultured in vitro is predictive for their growth and metastatic behavior in an orthotopic in vivo mouse model

Abstract Metastatic disease in solid cancer in general and specifically in colorectal cancer (CRC) is still the main reason for a poor prognosis underlining the need for new therapeutic strategies. Furthermore, a dysregulated secretion is one major driver of cancer progression and invasion and therefore holds promise as a general therapeutic target for the treatment of metastases. The overall aim of this study is to gain a quantitative understanding of the CRC secretome to elucidate the influence of the microenvironment on selected players and vice versa the influence of the secretome in vitro and in vivo. In a first attempt to elucidate the role of secreted factors in colon cancer progression, we cultured 20 CRC cell lines (five PDX derived and 15 commercial lines) in 3D spheroid cultures and compared the secreted cytokine profile with the 2D culture conditions of the same CRC cell line panel. We analyzed 45 cytokines in duplicates by using a human specific magnetic bead-based assay. Based on the cluster analysis of those cytokine profiles we categorized the CRC lines into low metastatic, intermediate and highly metastatic lines. To validate this clustering in vivo we implanted one representative cell line of each cluster orthotopically in immune-compromised NSG mice (n=10 per line in two independent experiments) and followed tumor growth by optical imaging twice a week over the course of up to 50 days. Tumor growth and metastases were confirmed by histological examination and terminal ex vivo imaging of the organs. The murine and human cytokine profile of different organs and the serum of tumor bearing mice was determined as well. The predicted highly metastatic cell line HCT116 displayed a latency of 24 days post implantation: after that time tumor cells were detected via optical imaging not only at the implantation site, but as well in kidney, spleen, liver and lungs in 100% of the examined animals. The intermediate cell line HT29 had a latency of 38 days and showed tumor growth in colon and liver of all animals and lung metastases on 80% of the implanted mice. Mice bearing the PDX derived cell line 269, representing the low metastatic phenotype, had to be sacrificed 50 days post implant. Tumor cells were detected at the implantation site. Micro-metastases in liver and lung were detected in only 75% of the animals. The secretome pattern specific for an individual cell line was predictive for the metastatic potential in vivo in an orthotopic mouse model. Of note, none of the mentioned cell lines metastasized when implanted subcutaneously. The detailed analysis of the mouse and human cytokine profiles will help to elucidate the complex crosstalk between tumor cells and the tumor microenvironment. The modulation of individual signaling cascades in vitro and in vivo will help to gain a deeper understanding of the metastatic process thereby enabling the development of possible new therapies. Citation Format: Jacqueline Bersano, Dorothee Lenhard, Kanstantsin Lashuk, Eva Oswald, Julia Schüler. The secretome of colorectal cancer cell lines cultured in vitro is predictive for their growth and metastatic behavior in an orthotopic in vivo mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3126.

Colorectal Liver Micrometastases: Association with RAS/TP53 Co-Mutation and Prognosis after Surgery.

Micrometastases, defined as microscopic cancer cells spatially separated from the macroscopically evident metastasis, are identified in 24% to 56% of resected colorectal liver metastases (CLMs). Somatic gene mutations have emerged as independent prognostic factors in CLM. This study aimed to determine the prognostic impact and risk factors for the presence of micrometastases, including somatic gene mutations. Prospective evaluation for micrometastases was performed at 2 centers in the US and France from 2015 to 2019. CLM specimens were cut radially from the tumor margin to surrounding grossly normal liver for a distance of 2 cm. Depending on CLM size, 3 to 8 specimens per patient were submitted for microscopic analysis. Somatic gene mutations were detected by next-generation sequencing. Among 140 patients undergoing CLM resection in the US (n = 84) and France (n = 56), 36 (26%) patients were found to have micrometastases. Five-year overall and recurrence-free survival rates with micrometastases were 39% and 0%, respectively, compared with 61% and 20% without micrometastases (both p < 0.05). In multivariable analyses, the presence of micrometastases was an independent risk factor for worse overall survival (hazard ratio 2.88, 95% CI 1.46 to 5.70, p = 0.002) and recurrence-free survival (hazard ratio 1.56, 95% CI 1.01 to 2.41, p = 0.046). In binary logistic regression analysis, RAS/TP53 co-mutation was found to significantly increase the risk of micrometastases (odds ratio 2.77, 95% CI 1.15 to 6.71, p = 0.024). Micrometastases are associated with significantly worse survival after CLM resection. RAS/TP53 co-mutation correlated with increased risk of micrometastases. Further studies are needed to determine strategies to eradicate micrometastases.

Non-invasive detection and complementary diagnosis of liver metastases via chemokine receptor 4 imaging

Noninvasive detection of early-stage liver metastases from different primary cancers is a pressing unmet medical need. The lack of both molecular biomarkers and the sensitive imaging methodology makes the detection challenging. In this study, we observed the elevated expression of chemokine receptor 4 (CXCR4) in uveal melanoma (UM) patient liver tissues, and high CXCR4 expression in liver metastases of UM murine models, regardless of the expression levels in the primary tumors. Based on these findings, we identified CXCR4 as an imaging biomarker and exploited a CXCR4-targeted MRI contrast agent ProCA32.CXCR4 for molecular MRI imaging. ProCA32.CXCR4 has strong CXCR4 binding affinity, high metal selectivity, and r1 and r2 relaxivities, which enables the sensitive detection of liver micrometastases. The MRI imaging capacity for detecting liver metastases was demonstrated in three UM models and one ovarian cancer model. The imaging results were validated by histological and immunohistochemical analysis. ProCA32.CXCR4 has strong potential clinical application for non-invasive diagnosis of liver metastases.

Mesoscale visualization of three-dimensional microvascular architecture and immunocyte distribution in intact mouse liver lobes.

Rational: The complex vascular architecture and diverse immune cells of the liver are critical for maintaining liver homeostasis. However, quantification of the network of liver vasculature and immunocytes at different scales from a single hepatic lobule to an intact liver lobe remains challenging.Methods: Here, we developed a fast and fluorescence-preserving transparency method, denoted liver-CUBIC, for systematic and integrated analysis of the microcirculation and the three-dimensional distribution of dendritic cells (DCs)/macrophages in intact liver lobes.Results: Whole-mount imaging at mesoscale revealed that the hepatic classical lobule preferred the oblate ellipsoid morphology in the mouse liver and exhibited hepatic sinusoids with heterogeneous arrangement and intricate loop structure. Liver fibrosis not only induces sinusoidal density increase but also promotes sinusoidal arrangement with increased sinusoidal branch and loop structure. Meanwhile, we found that CD11c+ DCs followed a lognormal distribution in the hepatic lobules, skewing toward lobular boundary in steady state. CCl4-induced chronic liver injury promoted CD11c+ DC rearrangement at the lobular border before the formation of liver fibrosis. Furthermore, through whole-mount imaging of tumor-immune cell-vascular crosstalk in intact lobes based on liver-CUBIC, we characterized an accumulation of CX3CR1+CCR2+F4/80+ macrophages at metastatic foci in early colorectal liver metastases. Importantly, colorectal cells secrete CCL2 to mobilize CX3CR1+CCR2+F4/80+ macrophages to accumulate at liver micrometastases, and the interruption of CCL2-induced macrophage accumulation inhibits early colonization of metastasis in the liver.Conclusions: Our investigation of the sinusoidal network and DC/macrophage arrangements through the liver-CUBIC approach and whole-mount imaging provide a powerful platform for understanding hepatic circulatory properties and immune surveillance in the liver.

Accuracy of Endoscopic Ultrasonography for Gastric Cancer Staging.

Gastric cancer remains a health problem, with treatment indications varying with the TNM stage. We aimed in this study to highlight the role of EUS in GC patients and also to calculate the accuracy, sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of EUS for T and N staging in our group of patients with this disease. In this study, we included 41 GC patients, and individual values for every T stage accuracy, sensitivity, specificity, PPV, NPV, correct staging, understaging, and overstaging were calculated. EUS overall accuracy for T staging was 58.53%, with the highest sensitivity reached for the T4 stage, 95.83%. For N+vs. N-staging, EUS accuracy was 68.29%, with a sensitivity of 75% and a specificity of 44.44%. The positive and negative predicted values for the presence or absence of nodal disease were 82.75%, respectively 33.33%. In conclusion, this study confirmed the importance of EUS for the assessment of GC T and N stage and highlighted the role of this tool in the detection of liver micrometastasis unrevealed by other imaging techniques like abdominal ultrasound or MSCT.

Inhibition of CCL7 derived from Mo-MDSCs prevents metastatic progression from latency in colorectal cancer

In colorectal cancer (CRC), overt metastases often appear after years of latency. But the signals that cause micro-metastatic cells to remain indolent, thereby enabling them to survive for extended periods of time, are unclear. Immunofluorescence and co-immunoprecipitation assays were used to explore the co-localization of CCL7 and CCR2. Immunohistochemical (IHC) assays were employed to detect the characters of metastatic HT29 cells in mice liver. Flow cytometry assays were performed to detect the immune cells. Bruberin vivo MS FX Pro Imager was used to observe the liver metastasis of CRC in mice. Quantitative real-time PCR (qRT-PCR) and western blot were employed to detect the expressions of related proteins. Trace RNA sequencing was employed to identify differentially expressed genes in MDSCs from liver micro-M and macro-M of CRC in mice. Here, we firstly constructed the vitro dormant cell models and metastatic dormant animal models of colorectal cancer. Then we found that myeloid-derived suppressor cells (MDSCs) were increased significantly from liver micro-metastases to macro-metastases of CRC in mice. Moreover, monocytic MDSCs (Mo-MDSC) significantly promoted the dormant activation of micro-metastatic cells compared to polymorphonuclear MDSCs (PMN-MDSC). Mechanistically, CCL7 secreted by Mo-MDSCs bound with membrane protein CCR2 of micro-metastatic cells and then stimulated the JAK/STAT3 pathway to activate the dormant cells. Low-dose administration of CCL7 and MDSCs inhibitors in vivo could significantly maintain the CRC metastatic cells dormant status for a long time to reduce metastasis or recurrence after radical operation. Clinically, the level of CCL7 in blood was positively related to the number of Mo-MDSCs in CCR patients, and highly linked with the short-time recurrence and distant metastasis. CCL7 secreted by Mo-MDSCs plays an important role in initiating the outgrowth of metastatic latent CRC cells. Inhibition of CCL7 might provide a potential therapeutic strategy for the prevention of metastasis recurrence.

Orthotopic Human Metastatic Uveal Melanoma Xenograft Mouse Models: Applications for Understanding the Pathophysiology and Therapeutic Management of Metastatic Uveal Melanoma

The propensity of uveal melanoma to metastasize to the liver hinders the accrual of micro-metastatic and end-stage disease tissue samples and restricts the investigation of metastatic uveal melanoma (MUM). Pre-clinical experimental animal models of MUM can help elucidate the pathophysiology of metastatic lesions and provide a tool for designing new therapeutic approaches for MUM. Here, we present an advanced model of hepatic metastases that enables quantitatively visualizing the development of individual hepatic tumor clones and estimating their growth kinetics and colonization efficiency. Similar to clinically observed liver metastases, these models enable the assessment of growth kinetics of the liver micro-metastases and the testing of therapeutic approaches for the treatment of MUM. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Experimental patient-derived xenograft mouse model of metastatic uveal melanoma Basic Protocol 2: Experimental liver micro-metastatic mouse model using splenic injection of metastatic uveal melanoma cells.

Partial hepatectomy enhances the growth of CC531 rat colorectal cancer cells both in vitro and in vivo

Partial hepatectomy (PHx) is the gold standard for the treatment of colorectal cancer liver metastases. However, after removing a substantial amount of hepatic tissue, growth factors are released to induce liver regeneration, which may promote the proliferation of liver micrometastases or circulating tumour cells still present in the patient. The aim of this study is to assess the effect of PHx on the growth of liver metastases induced by intrasplenic cell inoculation as well as on in vitro proliferation of the same cancer cell line. Liver tumours were induced in 18 WAG/RijHsd male rats, by seeding 250,000 syngeneic colorectal cancer cells (CC531) into the spleen. The left lateral lobe of the liver was mobilized and in half of the animals it was removed to achieve a 40% hepatectomy. Twenty-eight days after tumour induction, the animals were sacrificed and the liver was removed and sliced to assess the relative tumour surface area (RTSA%). CC531 cells were cultured in presence of foetal calf serum, non-hepatectomised (NRS) or hepatectomized rat serum (HRS), and their proliferation rate at 24, 48, and 72 h was measured. RTSA% was significantly higher in animals which had undergone PHx than in the controls (non-hepatectomised) (46.98 ± 8.76% vs. 18.73 ± 5.65%; p < 0.05). Analysing each lobe separately, this difference in favour of hepatectomized animals was relevant and statistically significant in the paramedian and caudate lobes. But in the right lobe the difference was scarce and not significant. In vitro, 2.5% HRS achieved stronger proliferative rates than the control cultures (10% FCS) or their equivalent of NRS. In this experimental model, a parallelism has been shown between the effect of PHx on the growth of colorectal cancer cells in the liver and the effect of the serum on those cells in vitro.

A hemi-spleen injection model of liver metastasis for prostate cancer.

Liver metastasis is not uncommon in men with metastatic castration-resistant prostate cancer (mCRPC), estimated at ~20% to 60% of advanced late-stage patients. Liver and other visceral metastases are associated with worse overall survival. Recent evidence suggests the frequency of visceral metastases may be increasing for reasons that are unclear but may be related to selective pressures induced by modern therapies, including second-generation antiandrogen receptor signaling inhibitors such as enzalutamide and abiraterone. Consequently, robust models to study the pathobiology of prostate cancer liver metastases and their response to therapy are urgently needed. Hemi-spleen injection of human (LN95, PC3, VCaP, and MDA-PCa-2b) or syngeneic (Myc-CaP) prostate cancer cells (1 × 106 ) was performed to seed liver metastases via the splenic vessels. Plasma levels of prostate-specific antigen (PSA) were monitored longitudinally in human androgen receptor-positive (AR+) models. Immunohistochemical staining of AR and HoxB13 was performed to document the prostatic origin of hepatic lesions. LN95, PC3, and Myc-CaP produced distinct liver micrometastases that progressed to macrometastases by ~2 to 4 weeks postinoculation, while inoculation of MDA-PCa-2b and VCaP only produced occasional micrometastases and seeding of individual cells adjacent to blood vessels, respectively, at the time points analyzed. All lesions are characterized by positive staining for nuclear AR and/or the prostate-specific differentiation marker HoxB13 depending on the model. Circulating PSA levels are strongly correlated with overall tumor burden in mice seeded with LN95. Histologic micrometastases and low levels of circulating PSA are detected in mice seeded with MDA-PCa-2b at ~60 days postinoculation, but no circulating PSA was detected in animals inoculated with VCaP up to ~75 days despite the presence of rare AR+ cells in the liver. The studies reported herein establish intrasplenic injection as a robust model of mCRPC liver metastasis. In addition, circulating PSA was validated as a noninvasive biomarker to longitudinally monitor overall tumor burden when using PSA+ models. Therefore, this model can be used to interrogate the pathophysiology of prostate cancer liver metastases, the microenvironmental factors permissive to such growth, immunologic variables, and the response of hepatic lesions to therapy.

Identification of primordial germ cell-like cells as liver metastasis initiating cells in mouse tumour models

Liver metastasis, characterized by the spread of tumors to the liver from other areas, represents a deadly disease with poor prognosis. Currently, there is no effective therapeutic strategies and/or agents to combat liver metastasis primarily due to the insufficient understanding of liver metastasis. To develop a promising strategy for targeting liver metastasis, understanding of a cell origin responsible for liver metastasis and how this cell can be pharmacologically eliminated are therefore crucial. Using diverse tumor models including p53−/− genetic mouse model and syngeneic tumor models, we identified primordial germ cell (PGC)-like tumor cells, which are enriched in earliest liver micro-metastasis (up to 99%), as a cell origin of liver metastasis. PGC-like tumor cells formed earliest micro-metastasis in liver and gradually differentiated into non-PGC-like tumor cells to constitute late macro-metastasis in the course of tumor metastasis. The liver metastasis-initiating cells (PGC-like tumor cells) display cell renewal and differentiation capabilities, resemble primordial germ cells (PGCs) in morphology and PGC marker gene expression, and express higher level of the genes linked to metastasis and immune escape compared with non-PGC-like tumor cells. Of note, Stellarhigh PGC-like tumor cells, but not Stellarlow non-PGC-like cells, sorted from primary tumors of p53−/− mice readily form liver metastasis. Depletion of PGC-like tumor cells through genetic depletion of any of key germ cell genes impairs liver metastasis, while increased PGC-like tumor cells by SMAD2 knockout is correlated with markedly enhanced liver metastasis. Finally, we present the proof of principle evidence that pharmacologically targeting BMP pathways serves as a promising strategy to eliminate PGC-like tumor cells leading to abrogating liver metastasis. Collectively, our study identifies PGC-like tumor cells as a cell origin of liver metastasis, whose depletion by genetically targeting core PGC developmental genes or pharmacologically inhibiting BMP pathways serves a promising strategy for targeting liver metastasis.

Abstract A97: Neutrophil extracellular traps (NETs) promote immune escape and metastatic growth after surgical stress

Abstract Introduction: Surgery induces local and systemic inflammatory and immunosuppressive disturbances that correlate with adverse cancer outcomes. Following surgical stress, NETs promote the growth of liver micrometastases. We hypothesize that NETs support metastatic growth by promoting immune escape. Methods: Liver ischemia reperfusion was performed 5 days after a murine liver metastatic model was established. Results: Liver PD-L1 expression increased and peaked between 6-48 hours after reperfusion. A smaller peak of PD-L1 was observed after 2 weeks as the metastatic tumors grew. During the first 48 hours, the predominant source of PD-L1 was CD45+-CD11b+-Ly6G+ cells. PD-L1 expression was significantly decreased when NETs were inhibited either genetically by using PAD4KO mice or DNAse injections. In vitro, we confirmed both human and murine NETs express high levels of PD-L1. After liver I/R, tumor-infiltrating T cells expressed inhibitory receptors and were highly dysfunctional with reduced cytokine production and proliferative capacity in the NET-rich tumors compared to tumors from sham mice. T-cell exhaustion was reversed in mice treated with DNAse or PAD4KO mice. Similar results were observed in vitro as NETs suppressed T-cell function via PDL-1/PD-1 interaction. Treating mice with anti-PD-L1 during the first 48 hours after reperfusion reversed T-cell exhaustion and decreased tumor growth. Conclusion: NETs induced by surgical stress cause immune escape by secreting PD-L1, increasing T-cell exhaustion, and upregulation of immunosuppressive ligands on tumor cells. These findings suggest avenues for overcoming the perioperative accelerated tumor growth and immunosuppressive state and ultimately improving long-term oncologic outcomes. Citation Format: Hamza O. Yazdani, Christof T. Kaltenmeier, David Geller, Samer Tohme. Neutrophil extracellular traps (NETs) promote immune escape and metastatic growth after surgical stress [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A97.

Neutrophil extracellular traps promote liver micrometastasis in pancreatic ductal adenocarcinoma via the activation of cancer‑associated fibroblasts.

Cancer‑associated fibroblasts (CAFs) promote the progression of pancreatic ductal adenocarcinoma (PDAC) via tumor‑stromal interactions. Neutrophil extracellular traps (NETs) are extracellular DNA meshworks released from neutrophils together with proteolytic enzymes against foreign pathogens. Emerging studies suggest their contribution to liver metastasis in several types of cancer. Herein, in order to investigate the role of NETs in liver metastasis in PDAC, the effects of NET inhibitors on spontaneous PDAC mouse models were evaluated. It was demonstrated that DNaseI, a NET inhibitor, suppressed liver metastasis. For further investigation, further attention was paid to liver micrometastasis and an experimental liver metastasis mouse model was used that was generated by intrasplenic tumor injection. Furthermore, DNaseI also suppressed liver micrometastasis and notably, CAFs accumulated in metastatic foci were significantly decreased in number. Invitro experiments revealed that pancreatic cancer cells induced NET formation and consequently NETs enhanced the migration of hepatic stellate cells, which was the possible origin of CAFs in liver metastasis. On the whole, these results suggest that NETs promote liver micrometastasis in PDAC via the activation of CAFs.

Expression of E-cadherin and specific CXCR3 isoforms impact each other in prostate cancer

BackgroundCarcinoma cells shift between epithelial and mesenchymal phenotypes during cancer progression, as defined by surface presentation of the cell-cell cohesion molecule E-cadherin, affecting dissemination, progression and therapy responsiveness. Concomitant with the loss of E-cadherin during the mesenchymal transition, the predominant receptor isoform for ELR-negative CXC ligands shifts from CXCR3-B to CXCR3-A which turns this classical G-protein coupled receptor from an inhibitor to an activator of cell migration, thus promoting tumor cell invasiveness. We proposed that CXCR3 was not just a coordinately changed receptor but actually a regulator of the cell phenotype.MethodsImmunoblotting, immunofluorescence, quantitative real-time PCR and flow cytometry assays investigated the expression of E-cadherin and CXCR3 isoforms. Intrasplenic inoculation of human prostate cancer (PCa) cells with spontaneous metastasis to the liver analyzed E-cadherin and CXCR3-B expression during cancer progression in vivo.ResultsWe found reciprocal regulation of E-cadherin and CXCR3 isoforms. E-cadherin surface expression promoted CXCR3-B presentation on the cell membrane, and to a lesser extent increased its mRNA and total protein levels. In turn, forced expression of CXCR3-A reduced E-cadherin expression level, whereas CXCR3-B increased E-cadherin in PCa. Meanwhile, a positive correlation of E-cadherin and CXCR3-B expression was found both in experimental PCa liver micro-metastases and patients’ tissue.ConclusionsCXCR3-B and E-cadherin positively correlated in vitro and in vivo in PCa cells and liver metastases, whereas CXCR3-A negatively regulated E-cadherin expression. These results suggest that CXCR3 isoforms may play important roles in cancer progression and dissemination via diametrically regulating tumor’s phenotype.

Metastasis of pancreatic cancer: An uninflamed liver micromilieu controls cell growth and cancer stem cell properties by oxidative phosphorylation in pancreatic ductal epithelial cells.

Pancreatic ductal adenocarcinoma (PDAC) is commonly diagnosed when liver metastases already emerged. We recently demonstrated that hepatic stromal cells determine the dormancy status along with cancer stem cell (CSC) properties of pancreatic ductal epithelial cells (PDECs) during metastasis. This study investigated the influence of the hepatic microenvironment - and its inflammatory status - on metabolic alterations and how these impact cell growth and CSC-characteristics of PDECs. Coculture with hepatic stellate cells (HSCs), simulating a physiological liver stroma, but not with hepatic myofibroblasts (HMFs) representing liver inflammation promoted expression of Succinate Dehydrogenase subunit B (SDHB) and an oxidative metabolism along with a quiescent phenotype in PDECs. SiRNA-mediated SDHB knockdown increased cell growth and CSC-properties. Moreover, liver micrometastases of tumor bearing KPC mice strongly expressed SDHB while expression of the CSC-marker Nestin was exclusively found in macrometastases. Consistently, RNA-sequencing and in silico modeling revealed significantly altered metabolic fluxes and enhanced SDH activity predominantly in premalignant PDECs in the presence of HSC compared to HMF. Overall, these data emphasize that the hepatic microenvironment determines the metabolism of disseminated PDECs thereby controlling cell growth and CSC-properties during liver metastasis.

The antimetastatic effect and underlying mechanisms of thioredoxin reductase inhibitor ethaselen

Treating cancer metastasis is of vital importance to prolong patients’ survival. Thioredoxin reductase (TrxR) is overexpressed in many cancer types and has been recognized as an anti-cancer target. The organoselenium compound ethaselen (BBSKE) has been proved to be a TrxR inhibitor and inhibit various types of tumor growth. However, whether BBSKE could inhibit tumor metastasis remains unclear. In this study, we aim to explore the antimetastatic effect of BBSKE and underlying mechanisms. BBSKE was found to dose-dependently suppress migration and invasion of MCF-7 and LoVo cells in vitro. The underlying mechanisms may include inhibition of TrxR activity, elevation of reactive oxygen species (ROS), decrease of EGFR activation and HER2 expression. Besides, the epithelial to mesenchymal transition process and expression of CD44, MMP-9, VEGFR2 and PD-L1 were also abrogated. Decreased migration and invasion, lower expression levels of EGFR, HER2, N-cadherin, CD44, MMP-9, VEGFR2 and PD-L1 were also observed in TrxR1-knockdown MCF-7 and LoVo cells. In the mouse breast cancer 4T1 model, BBSKE not only inhibited progression of primary tumor, but also suppressed formation of metastatic lung nodules and liver micro-metastases, indicating that BBSKE could effectively abolish tumor metastasis. In conclusion, our findings show that BBSKE is able to inhibit migration and invasion of cancer cells in vitro and in vivo, and may be used to prevent and treat metastasis.

Resection of Hepatic Lesions Perfused by the Cholecystic Vein Using Indocyanine Green Navigation in Patients with cT2 Gallbladder Cancer.

Various approaches to hepatectomy have been proposed for cT2 gallbladder cancers (GBC), but the optimal management strategy remains unclear. The aim of this study is to assess the effectiveness of using an indocyanine green (ICG)-based intraoperative navigation system during hepatic resection for cT2 GBC. From September 2007 to December 2017, 24 consecutive patients diagnosed with cT2 GBC underwent hepatic resection using ICG navigation. After cannulation of the cholecystic artery, ICG diluted with dissolution liquid was injected and ICG fluorescence illumination was visualized with the HyperEye Medical System. And additional histopathological examination was performed on the most recent 15 of the 24 patients for detection of microscopic liver metastasis. For all patients, the disease-free survival rate was 59.1% at 5years and overall survival rate was 86.2% at 5years. Microscopic liver metastasis was detected in the resected liver in 3 (20%) of 15 patients, whose site of liver was S6, S5, and S5, respectively. The weight of the liver resected using ICG navigation was significantly smaller than that of S4a/S5 segmentectomy (P < 0.0001). Resected hepatic lesion using ICG imaging was possible to perform hepatectomy including liver micro-metastasis without excess or deficiency. This procedure might be novel intraoperative imaging method to provide valuable information on the optimal surgical approach to cT2 GBC.