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Vessel co-option correlates with resistance against anti-angiogenic agents and chemotherapy in colorectal cancer liver metastasis (CRCLM). We previously identified higher intensity of neutrophils in the tumour microenvironment of vessel co-opting CRCLM lesions compared to their angiogenic counterparts. Herein, we demonstrated that over 50% of the neutrophils in vessel co-opting lesions are expressing pro-apoptotic markers including cleaved caspase-3 and poly (ADP-ribose) polymerase-1 (PARP-1). Our previous publications suggested upregulation of transforming growth factor-beta (TGFβ1) in the microenvironment of vessel co-option CRCLM. Therefore, we examined the effect of TGFβ1 on the expression of cleaved caspase-3 and PARP-1 in neutrophils in vitro. Significantly, we noticed the upregulation of pro-apoptotic markers upon exposure to TGFβ1. This finding might pave the way to determine the role of neutrophils in developing vessel co-option in CRCLM in the future.

Colorectal cancer liver metastasis (CRCLM) is one of the deadliest cancers. CRCLM tumours have two distinct histopathological growth patterns (HGPs) including desmoplastic HGP (DHGP) and replacement HGP (RHGP). The DHGP tumours are angiogenic, while their RHGP counterparts are vessel co-opting. The patients with DHGP tumours have a better response to anti-angiogenic agents and chemotherapy, as well as the prognosis. To determine the influence of vitamin D supplementation in CRCLM, we analyzed the HGPs and the 5-year OS of CRCLM patients (n=106). Interestingly, we found an inverse correlation between vitamin D supplementation and the presence of RHGP tumours in CRCLM patients. Additionally, the 5-year OS of the patients that administered vitamin D was significantly higher. The cancer cells in RHGP lesions are characterized by direct contact with the hepatocytes, and this phenomenon enhances the motility of the cancer cells and facilitates their infiltration through liver parenchyma to co-opt the pre-existing vessels. Significantly, our in vitro data demonstrated the downregulation of motility markers in the co-cultured cancer cells with hepatocytes upon exposure to vitamin D. Altogether, this study highlights the role of vitamin D in CRCLM and provides a rationale to investigate the contribution of vitamin D supplementation to the prognosis of CRCLM patients.

Introduction: Colorectal cancer liver metastasis (CRCLM) remains a lethal diagnosis with an overall 5-year survival rate of 5–10%. Two distinct histopathological growth patterns (HGPs) of CRCLM are known to have significantly differing rates of patients survival, and response to treatment. We set out to review the results of 275 patients who underwent liver resection for CRCLM at the McGill University Health Center (MUHC) and analyze their clinical outcome, mutational burden and pattern of cancer progression in light of their HGP’s, and to consider their potential effect on surgical decision making. Methods We performed a retrospective multivariate analysis on clinical data from patients with CRCLM (n = 275) who underwent liver resection at the McGill University Health Center (MUHC). All tumors were scored using international consensus guidelines by pathologists trained in HGP scoring. Results 109 patients (42.2%) were classified as desmoplastic and angiogenic whereas 149 patients (57.7%) were non-desmoplastic and vessel co-opting. The 5 year survival rates for angiogenic patients compared to vessel co-opting patients was 47.1% and 13% respectively (p

Colorectal cancer liver metastatic (CRCLM) tumours present as two main histopathological growth patterns (HGPs) including desmoplastic HGP (DHGP) and replacement HGP (RHGP). The DHGP tumours obtain their blood supply by sprouting angiogenesis, whereas the RHGP tumours utilize an alternative vascularisation known as vessel co-option. In vessel co-option, the cancer cells hijack the mature sinusoidal vessels to obtain blood supply. Vessel co-option has been reported as an acquired mechanism of resistance to anti-angiogenic treatment in CRCLM. Here, we show the connection between the concentration of serum cholesterol and the development of vessel co-option in CRCLM. Our clinical data suggested that the elevation of serum cholesterol levels correlates with the risk of developing vessel co-opting tumours. Moreover, inhibition of the key modulators of cholesterol metabolism including HMGCR or PCSK9 attenuated the development of CRCLM tumours, as well as vessel co-option in vivo. Altogether, our data uncovered the importance of cholesterol in the development of vessel co-option tumours and demonstrated PCSK9 and HMGCR inhibitors as promising strategies to mitigate the development of vessel co-option tumours in CRCLM.

Colorectal cancer liver metastasis (CRCLM) is one of the deadliest cancers. CRCLM tumours have two distinct histopathological growth patterns (HGPs) including desmoplastic HGP (DHGP) and replacement HGP (RHGP). The DHGP tumours are angiogenic, while their RHGP counterparts are vessel co-opting. The patients with RHGP tumours showed poor response to anti-angiogenic agents, as well as a worse prognosis. Herein, we conducted a retrospective cohort study that comprised 106 CRCLM patients to examine the effect of vitamin D supplementation on the HGPs of the tumours and the 5-year overall survival (OS). Interestingly, we found an inverse correlation between vitamin D supplementation and the presence of RHGP tumours in CRCLM patients. Additionally, the population who used vitamin D supplementation had significantly better 5-year OS. Moreover, our in vivo results suggested that vitamin D supplementation significantly improves the response of CRCLM tumours to anti-angiogenic therapy. Mechanistically, we found that vitamin D can suppress cancer cell motility, which is essential for the development of vessel co-option tumours and resistance to anti-angiogenic therapy. Collectively, this study suggests vitamin D supplementation is a promising way to attenuate resistance to anti-angiogenic therapy and improve the prognosis of CRCLM patients. Citation Format: Miran Rada, Lucyna Krzywon, Audrey Kapelanski-Lamoureux, Stephanie Petrillo, Anthoula Lazaris, Peter Metrakos. Vitamin D supplementation attenuates resistance to anti-angiogenic therapy in colorectal cancer liver metastases. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4601.

Colorectal cancer liver metastatic (CRCLM) tumours present as two main histopathological growth patterns (HGPs) including desmoplastic HGP (DHGP) and replacement HGP (RHGP). The DHGP tumours obtain their blood supply by sprouting angiogenesis, whereas the RHGP tumours utilize an alternative vascularisation known as vessel co-option. In vessel co-option, the cancer cells hijack the mature sinusoidal vessels to obtain blood supply. Vessel co-option has been reported as an acquired mechanism of resistance to anti-angiogenic treatment in CRCLM. Herein, we show the connection between serum cholesterol concentration and vessel co-option development in CRCLM. Our clinical data suggested that the elevation of serum cholesterol levels correlates with the risk of developing vessel co-opting tumours. Moreover, inhibition of PCSK9, the key modulator of cholesterol metabolism, significantly attenuated the development of vessel co-opting CRCLM tumours and sensitized the tumours to anti-angiogenic therapy in vivo. Altogether, these data suggest the importance of cholesterol in the development of vessel co-option tumours and propose that inhibiting PCSK9 is a promising strategy to overcome resistance to anti-angiogenic therapy in CRCLM. Citation Format: Miran Rada, Lucyna Krzywon, Audrey Kapelanski-Lamoureux, Stephanie Petrillo, Andrew Reynolds, Anthoula Lazaris, Nabil Seidah, Peter Metrakos. Inhibition of pcsk9 impairs the development of vessel co-option and potentiates anti-angiogenic therapy in colorectal cancer liver metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1741.

Colorectal cancer liver metastases (CRCLM) that present with a replacement histopathological growth pattern (HGP) are resistant to neoadjuvant anti-angiogenic therapy. Surrogate biomarkers are not available to preoperatively identify patients with these tumors. Here we identify differentially expressed genes between CRCLM with a replacement HGP and those with a desmoplastic HGP using RNA sequencing. We demonstrate that LOXL4 is transcriptionally upregulated in replacement HGP CRCLM compared with desmoplastic HGP CRCLM and the adjacent normal liver. Interestingly, lysyl oxidase-like 4 (LOXL4) protein was expressed by neutrophils present in the tumor microenvironment in replacement HGP CRCLM. We further demonstrate that LOXL4 expression is higher in circulating neutrophils of cancer patients compared with healthy control patients and its expression can be induced by stimulation with lipopolysaccharide and TNF-α. Our study is the first to show the expression of LOXL4 in neutrophils and reveals the potential for LOXL4-expressing neutrophils to support the replacement HGP phenotype and to serve as a surrogate biomarker for this subtype of CRCLM. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Objective The liver is regularly exposed to changing metabolic and inflammatory environments. It must sense and adapt to metabolic need while balancing resources required to protect itself from insult. Peroxisome proliferator activated receptor gamma coactivator-1 alpha (PGC-1α) is a transcriptional coactivator expressed as multiple, alternatively spliced variants transcribed from different promoters that coordinate metabolic adaptation and protect against inflammation. It is not known how PGC-1α integrates extracellular signals to balance metabolic and anti-inflammatory outcomes. Methods Primary mouse hepatocytes were used to evaluate the role(s) of different PGC-1α proteins in regulating hepatic metabolism and inflammatory signaling downstream of tumor necrosis factor alpha (TNFα). Gene expression and signaling analysis were combined with biochemical measurement of apoptosis using gain- and loss-of-function in vitro and in vivo. Results Hepatocytes expressed multiple isoforms of PGC-1α, including PGC-1α4, which microarray analysis showed had common and isoform-specific functions linked to metabolism and inflammation compared with canonical PGC-1α1. Whereas PGC-1α1 primarily impacted gene programs of nutrient metabolism and mitochondrial biology, TNFα signaling showed several pathways related to innate immunity and cell death downstream of PGC-1α4. Gain- and loss-of-function models illustrated that PGC-1α4 uniquely enhanced expression of anti-apoptotic gene programs and attenuated hepatocyte apoptosis in response to TNFα or lipopolysaccharide (LPS). This was in contrast to PGC-1α1, which decreased the expression of a wide inflammatory gene network but did not prevent hepatocyte death in response to cytokines. Conclusions PGC-1α variants have distinct, yet complementary roles in hepatic responses to metabolism and inflammation, and we identify PGC-1α4 as an important mitigator of apoptosis., Graphical abstract Image 1, Highlights • Multiple isoforms of PGC-1α are expressed in hepatocytes, including PGC-1α4. • PGC-1α1 and PGC-1α4 share many metabolic targets, but PGC-1α4 has unique functions linked to hepatic inflammatory signalling. • PGC-1α4 attenuates hepatocyte apoptosis in response to TNFα and LPS in vitro and in vivo. • Inflammatory signaling influences PGC-1α4 localization in hepatocytes.

Claudin-2 promotes breast cancer liver metastasis by enabling seeding and early cancer cell survival. We now demonstrate that Claudin-2 is functionally required for colorectal cancer liver metastasis and that Claudin-2 expression in primary colorectal cancers is associated with poor overall and liver metastasis-free survival. We have examined the role of Claudin-2, and other claudin family members, as potential prognostic biomarkers of the desmoplastic and replacement histopathological growth pattern associated with colorectal cancer liver metastases. Immunohistochemical analysis revealed higher Claudin-2 levels in replacement type metastases when compared to those with desmoplastic features. In contrast, Claudin-8 was highly expressed in desmoplastic colorectal cancer liver metastases. Similar observations were made following immunohistochemical staining of patient-derived xenografts (PDXs) that we have established, which faithfully retain the histopathology of desmoplastic or replacement type colorectal cancer liver metastases. We provide evidence that Claudin-2 status in patient-derived extracellular vesicles may serve as a relevant prognostic biomarker to predict whether colorectal cancer patients have developed replacement type liver metastases. Such a biomarker will be a valuable tool in designing optimal treatment strategies to better manage patients with colorectal cancer liver metastases., Tabariès et al. describe that claudin 2 is a promoter of colorectal cancer liver metastasis. Furthermore, high Claudin-2 expression is associated with shorter time to liver-specific recurrence and is a biomarker of replacement type CRC liver metastases.

BACKGROUND Different histological growth patterns (HGPs) of colorectal carcinoma (CRC) liver metastasis are associated with patients’ prognosis and response to antiangiogenic therapy. However, the relationship between HGPs of liver metastasis and clinicopathological and genomic characteristics of primary cancer has not been well established. AIM To assess whether certain clinicopathological and genomic features of primary CRC could predict the HGPs of liver metastasis. METHODS A total of 29 patients with paired resections of both primary CRC and liver metastasis were divided into two groups: A (15 cases with desmoplastic liver metastasis) and B (14 cases with replacement liver metastasis). Clinical information was obtained from patients’ charts. Mismatch repair proteins, BRAFV600E, and PD-L1 were evaluated by immunohistochemistry. Five cases were selected randomly from each group for whole exome sequencing (WES) analysis. RESULTS In the primary tumor, expanding growth pattern, low tumor budding score (TBS), and Crohn’s disease-like response (CDR) were associated with desmoplastic liver metastasis and better overall survival, whereas infiltrating growth pattern alone of primary carcinoma could predict the replacement liver metastasis and worse overall survival (P < 0.05). On WES analysis, primary carcinoma with desmoplastic liver metastasis showed mutations in APC (4/5); TP53 (3/5); KRAS, PIK3CA, and FAT4 (2/5); BRCA-1, BRCA2, BRAF, and DNAH5 (1/5), whereas primary carcinoma with replacement liver metastasis showed mutations in APC and TP53 (3/5); KRAS, FAT4, DNH5, SMAD, ERBB2, ERBB3, LRP1, and SDK1 (1/5). CONCLUSION The HGPs, TBS, and CDR of primary CRC as well as the presence of specific genetic mutations such as those in PIK3CA could be used to predict the HGPs of liver metastasis, response to therapy, and patients’ prognosis.

Resistance to anti-angiogenic therapy is a major challenge in the treatment of colorectal cancer liver metastases (CRCLMs). Vessel co-option has been identified as a key contributor to anti-angiogenic therapy resistance in CRCLMs. Recently, we identified a positive correlation between the expression of Angiopoietin1 (Ang1) in the liver and the development of vessel co-opting CRCLM lesions in vivo. However, the mechanisms underlying its stimulation of vessel co-option are unclear. Herein, we demonstrated Ang1 as a positive regulator of actin-related protein 2/3 (ARP2/3) expression in cancer cells, in vitro and in vivo, which is known to be essential for the formation of vessel co-option in CRCLM. Significantly, Ang1-dependent ARP2/3 expression was impaired in the cancer cells upon Tie2 or PI3K/AKT inhibition in vitro. Taken together, our results suggest novel mechanisms by which Ang1 confers the development of vessel co-option in CRCLM, which, targeting this pathway, may serve as promising therapeutic targets to overcome the development of vessel co-option in CRCLM.

Vessel co-option is correlated with resistance against anti-angiogenic therapy in colorectal cancer liver metastases (CRCLM). Vessel co-opting lesions are characterized by highly motile cancer cells that move toward and along the pre-existing vessels in the surrounding nonmalignant tissue and co-opt them to gain access to nutrients. To access the sinusoidal vessels, the cancer cells in vessel co-opting lesions must displace the hepatocytes and occupy their space. However, the mechanisms underlying this displacement are unknown. Herein, we examined the involvement of apoptosis, autophagy, motility, and epithelial–mesenchymal transition (EMT) pathways in hepatocyte displacement by cancer cells. We demonstrate that cancer cells induce the expression of the proteins that are associated with the upregulation of apoptosis, motility, and EMT in adjacent hepatocytes in vitro and in vivo. Accordingly, we observe the upregulation of cleaved caspase-3, cleaved poly (ADP-ribose) polymerase-1 (PARP-1) and actin-related protein 2/3 (ARP2/3) in adjacent hepatocytes to cancer cell nests, while we notice a downregulation of E-cadherin. Importantly, the knockdown of runt-related transcription factor 1 (RUNX1) in cancer cells attenuates the function of cancer cells in hepatocytes alterations in vitro and in vivo. Altogether, our data suggest that cancer cells exploit various mechanisms to displace hepatocytes and access the sinusoidal vessels to establish vessel co-option.

Translocator protein (TSPO, 18kDa) levels increase in parallel with the evolution of simple steatosis (SS) to nonalcoholic steatohepatitis (NASH) in non-alcoholic fatty liver disease (NAFLD). However, TSPO function in SS and NASH is unknown. Loss of TSPO in hepatocytes in vitro downregulated acetyl-CoA acetyltransferase 2 (ACAT2) and increased free cholesterol (FC). FC accumulation induced endoplasmic reticulum stress via IRE1A and PERK/ATF4/CHOP pathways and autophagy. TSPO deficiency activated cellular adaptive antioxidant protection; this adaptation was lost upon excessive FC accumulation. 19-Atriol, a TSPO ligand, blocked cholesterol binding and recapitulated many of the alterations seen in TSPO-deficient cells. These data suggest that TSPO deficiency accelerated the progression of SS. In NASH, however, loss of TSPO ameliorated liver fibrosis through downregulation of bile acid synthesis by reducing CYP7A1 and CYP27A1 levels and increasing farnesoid X receptor expression. These studies indicate a dynamic and complex role for TSPO in the evolution of NAFLD.

Vessel co-option in colorectal cancer liver metastases (CRCLM) has been recognized as one of the mechanistic pathways of resistance against anti-angiogenic therapy. The cancer cells are highly motile in co-opted lesions, which move toward and along the pre-existing sinusoidal vessels and hijack them to gain access to nutrient. The movement of cancer cells is accompanied by displacement of the hepatocytes. However, the molecular mechanisms underlying this displacement are unclear yet. To examine whether apoptosis involved in hepatocytes displacement by cancer cells in co-opted lesions, we performed immunohistochemical staining for pro-apoptotic markers, such as cleaved caspase-3 and cleaved PARP-1. We observed overexpression of pro-apoptotic markers in liver parenchyma of co-opted lesions compared to angiogenic lesions, specifically the hepatocytes that are in close proximity to the cancer cells. In vitro, we found that culturing hepatocytes with either colorectal cancer cells or conditioned media of co-opted CRCLM organoids induces apoptosis. Importantly, our results also suggested proprotein convertase subtilisin/kexin type 9 (PCSK-9 or PC-9) as a potential mediator of cancer cells-driven hepatocytes apoptosis. Altogether, these results confirm that cancer cells exploit apoptosis to establish vessel co-option in CRCLM. Citation Format: Miran Rada, Migmar Tsamchoe, Audrey Kapelanski-Lamoureux, Jessica Bloom, Stephanie Petrillo, Sébastien Tabariès, Diane H. Kim, Peter Younan, Alex Gregorieff, Peter Siegel, Anthoula Lazaris, Peter Metrakos. Cancer cells induce apoptosis in hepatocytes as one of the mechanisms to displace hepatocytes in vessel co-opted colorectal cancer liver metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1927.

Summary Translocator protein (TSPO, 18 kDa) levels increase in parallel with the evolution of simple steatosis (SS) to nonalcoholic steatohepatitis (NASH) in nonalcoholic fatty liver disease (NAFLD). However, TSPO function in SS and NASH is unknown. Loss of TSPO in hepatocytes in vitro downregulated acetyl-CoA acetyltransferase 2 and increased free cholesterol (FC). FC accumulation induced endoplasmic reticulum stress via IRE1A and protein kinase RNA-like ER kinase/ATF4/CCAAT-enhancer-binding protein homologous protein pathways and autophagy. TSPO deficiency activated cellular adaptive antioxidant protection; this adaptation was lost upon excessive FC accumulation. A TSPO ligand 19-Atriol blocked cholesterol binding and recapitulated many of the alterations seen in TSPO-deficient cells. These data suggest that TSPO deficiency accelerated the progression of SS. In NASH, however, loss of TSPO ameliorated liver fibrosis through downregulation of bile acid synthesis by reducing CYP7A1 and CYP27A1 levels and increasing farnesoid X receptor expression. These studies indicate a dynamic and complex role for TSPO in the evolution of NAFLD., Graphical abstract, Highlights • TSPO expression levels correlate with the progression of NAFLD • TSPO deficiency inhibits ACAT2 leading to FC accumulation • Loss of TSPO in hepatocytes leads to FC accumulation that promotes simple steatosis • Loss of TSPO attenuates liver fibrosis via downregulation of bile acid production, Molecular biology; Cell biology; Metabolomics

Polymorphisms and decreased activity of methylenetetrahydrofolate reductase (MTHFR) are linked to disease, including cancer. However, epigenetic regulation has not been thoroughly studied. Our goal was to generate DNA methylation profiles of murine/human MTHFR gene regions and examine methylation in brain and liver tumors. Pyrosequencing in four murine tissues revealed minimal DNA methylation in the CpG island. Higher methylation was seen in liver or intestine in the CpG island shore 5' to the upstream translational start site or in another region 3' to the downstream start site. In the latter region, there was negative correlation between expression and methylation. Three orthologous regions were investigated in human MTHFR, as well as a fourth region between the two translation start sites. We found significantly increased methylation in three regions (not the CpG island) in pediatric astrocytomas compared with control brain, with decreased expression in tumors. Methylation in hepatic carcinomas was also increased in the three regions compared with normal liver, but the difference was significant for only one CpG. This work, the first overview of the Mthfr/MTHFR epigenetic landscape, suggests regulation through methylation in some regions, demonstrates increased methylation/decreased expression in pediatric astrocytomas, and should serve as a resource for future epigenetic studies.

Colorectal cancer liver metastasis (CRCLM) has two major morphological growth pattern subtypes including desmoplastic (DHGP) and replacement (RHGP). The DHGP tumors depend on angiogenesis for their growth. Conversely, RHGP tumors are non-angiogenic and obtain their blood supply through vessel co-option, which the cancer cells hijacking pre-existing blood vessels of the surrounding liver tissue. Therefore, anti-angiogenic therapies have shown modest efficacy in CRCLM patients with RHGP lesions. In this study, we found overexpression of Runt Related Transcription Factor-1 (RUNX1) in the cancer cells of RHGP lesions. Upregulation of RUNX1 is positively correlated with cancer cell motility and epithelial mesenchymal transition (EMT) in vitro. Our results also showed that RUNX1 regulated by Transforming Growth Factor Beta-1 (TGFβ1) through TGFβ Receptor II (TGFβRII). Significantly, RUNX1 knockdown induced conversion RHGP lesions to DHGP in vivo. Collectively, this study suggests RUNX1 as a potential target therapy to overcome resistance to anti-angiogenic therapy in CRCLM. Citation Format: Miran Rada, Audrey Kapelanski-Lamoureux, Stephanie Petrillo, Sébastien Tabariès, Peter Siegel, Anthoula Lazaris, Peter Metrakos. Runt related transcription factor-1 (runx1) plays a central role in vessel co-option of colorectal cancer liver metastases [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3935.

Introduction: Prostate cancer (PCa) is the second most frequent cancer in men and a leading cause of cancer-related mortality. Despite major advances in immunotherapy, PCa remains a poor responder. Metastatic PCa is responsible for the majority of PCa-associated mortality. Most PCa metastases are multifocal and display a strong bones tropism (91.1% of cases), but PCa metastases can also spread to the lymph nodes (8.7%), lungs (5.7%), liver (4.5%) and brain (1.8%). Liver metastases are associated with worse prognosis but due to their multifocal nature and frequent spreading to other sites, PCa metastases are rarely resected. Therefore, immunologic characterization of these lesions concomitant with generation of research tools derived from these lesions are urgently needed to understand how to intercept disease progression. Methods: A 62-year-old male who previously underwent radical prostatectomy in 2016 was diagnosed in July 2018 with a single liver metastasis (5.3 cm) by MRI. The tumor was surgically resected and tumor tissue along with peripheral blood was collected and processed for in-depth immunologic/molecular characterization and generation of tumor models. The study was done in accordance with the guidelines approved by MUHC IRB. Prior written informed consent was obtained from the subject to participate in the study (protocol: SDR-11-066). Results: The prostatic origin of the tumor mass was confirmed by positivity for PSMA and NKX3.1 expression. Patient-derived xenografts, 2D cell and organoid cultures were generated and immunophenotyping of the innate and adaptive peripheral and tumor-infiltrating immune cells subsets was performed. Genomic alterations are currently being characterized by multiplex ligation-dependent probe amplification (MLPA). Additionally, chromatin accessibility-based characterization of the gene regulatory network of tumor luminal cells (CD49-CD26+) using the assay for transposase-accessible chromatin using sequencing (ATAC-seq) together with RNA-seq is presently under way. Conclusions: Our collaborative effort will provide the much-needed research tools required to model and understand the processes leading to the rare, but lethal, progression from a localized PCa lesion to liver metastases. Combined with other ongoing research efforts, we believe this case will help us understand the molecular basis to the liver tropism of a subset of PCa metastases and ultimately provide biomarkers for early identification of patients with increased metastatic potential as well as a basis to determine the appropriate immunotherapy modality for metastatic patients. Citation Format: Aurélie Y. Le Page, Anna de Polo, K-P Guérard, A. Lazaris, S.K. Petrillo, W. Ebrahimizadeh, S. Tabariès, S. Shinde-Jadhav, A. Feldiorean, N. Boufaeid, W. Kassouf, C. Piccirillo, P.M. Siegel, A. Aprikian, A. Gregorieff, J. Lapointe, P. Metrakos, D.P. Labbé. Immune profiling and organoids generation of a rare case of prostate cancer liver metastasis [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A26.

Colorectal cancer liver metastases (CRCLM) that receive their blood supply via vessel co-option are associated with a poor response to anti-angiogenic therapy. Angiopoietins (Ang1 and Ang2) with their Tyrosine-protein kinase receptor (Tie2) have been shown to support vessel co-option. We demonstrate significantly higher expression of Ang1 in hepatocytes adjacent to the tumor region of human chemona&iuml, ve and treated co-opting (replacement histopathological growth patterns: RHGP) tumors. To investigate the role of the host Ang1 expression, Ang1 knockout (KO) mice were injected intra-splenically with metastatic MC-38 colon cancer cells that develop co-opting liver metastases. We observed a reduction in the number of liver metastases and interestingly, for the first time, the development of angiogenic driven desmoplastic (DHGP) liver metastases. In addition, in-vitro, knockout of Ang1 in primary hepatocytes inhibited viability, migration and invasion ability of MC-38 cells. We also demonstrate that Ang 1 alone promotes the migration and growth of both human and mouse colon cancer cell lines These results provide evidence that high expression of Ang1 in the host liver is important to support vessel co-option (RHGP lesions) and when inhibited, favours the formation of angiogenic driven liver metastases (DHGP lesions).

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Three different histopathological growth patterns (HGP) have been identified in colorectal cancer liver metastases (CRCLM) resected from patients: the desmoplastic HGP, the pushing HGP, and the replacement HGP. Evidence suggests that the predominant growth pattern with which a CRCLM presents has clinically relevant prognostic implications. We have shown that patients with replacement HGP lesions who received bevacizumab plus chemotherapy had a worse pathological response and five-year overall survival than those with desmoplastic HGP lesions receiving the same treatment. We have also demonstrated that CRCLM with the replacement HGP promote vessel co-option for vascularization, rather than sprouting angiogenesis as seen in desmoplastic HGP metastases. These findings point to the growth patterns in CRCLM possibly serving as predictive biomarkers of response to anti-angiogenic therapy, when no such biomarker has been validated to date. However, HGP scoring is performed on resected liver metastatic tissue, implying that preoperative treatment precedes growth pattern assessment. Therefore, surrogate markers for the HGPs that can be appraised prior to surgery would be helpful to inform clinical decision-making about whether a patient with CRCLM may benefit from anti-angiogenic treatment. This project aims to identify genes that are differentially expressed between CRCLM presenting with either the replacement or desmoplastic HGP. RNA sequencing (RNA-Seq) was used to compare the transcriptional profiles of these distinct CRCLM. A gene expression signature was generated from the RNA-Seq data to include genes that were upregulated in the replacement HGP liver metastases. Immunostaining of select genes from this signature was performed to validate these findings on human tumor tissue. Initial analysis of our RNA-Seq data identified 525 genes that are differentially expressed between the replacement and desmoplastic HGP CRCLM, of which 53 genes met the criteria for the gene expression signature. Pathway analysis of these genes identified pathways involved in the immune system and extracellular matrix (ECM) to be upregulated in the replacement HGP lesions. Subsequent immunostaining revealed the expression pattern of lysyl oxidase like-4 (LOXL4) protein to be distinct between the HGPs - significantly greater quantities of LOXL4-expressing neutrophils were detected in the replacement HGP tumor microenvironment. Characterizing differences in gene expression between replacement and desmoplastic HGP CRCLM is expected to provide some insight into the mechanisms responsible for generating these distinct growth patterns. Our findings suggest that further investigations into the role of tumor-associated leukocytes and ECM remodelling may ultimately lead to the development of biomarkers and new therapeutic targets for replacement HGP CRCLM. Citation Format: Vincent Palmieri, Anthoula Lazaris, Stephanie Petrillo, Hussam Alamri, Abdellatif Amri, Woong-Yang Park, Zu-hua Gao, Peter Metrakos. LOXL4-expressing neutrophils are found in colorectal cancer liver metastases resistant to anti-angiogenic therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2777.

Colorectal carcinoma (CRC) remains the second leading cause of cancer death in the western world. Over 50% of CRC patients develop liver metastases (LM) and 90% will succumb to their disease. Liver resection of the LMs provides the only possibility of cure, but only 20% of colorectal cancer liver metastases (CRCLM) patients are resectable. The combination of angiogenic inhibitors (AI: anti-VEGF) with chemotherapy is the current form of treatment. Unfortunately, 65-70% of the patients continue on chemotherapy until resistance develops and then are treated with second, third and some times a fourth line of treatment, with an expected median overall survival of 24-28 months. We have no way of identifying those CRCLM patients that would respond/benefit to the addition of anti-angiogenic therapies (e.g. Bevacizumab). Recently we have identified two CRCLM histologic growth patterns (HGP) that predict treatment response and survival: 1) Desmoplastic (DHGP), a desmoplastic ring separating cancer cells from the liver parenchyma and lesions grow by angiogenesis; 2) Replacement or infiltrative (RHGP), tumor cells infiltrate the parenchymal cells in the liver as the lesions grow by co-opting the sinusoidal blood vessels between the liver cell plates. We showed that CRCLM patients with predominantly desmoplastic HGP metastasis receiving AIs plus chemotherapy have more than double the 5-year overall survival compared to patients with replacement HGP who have received the same treatment. In addition, our clinical data revealed that Angiogenic Inhibitors could negatively affect outcomes in patients with replacement HGPs. These non-angiogenic lesions do not respond to angiogenic inhibitors. To further our understanding of the molecular differences between the two HGPs we demonstrated by knocking out ARPC3 (Actin-related protein 2/3 complex subunit 3, involved in actin polymerization) in the human colon cancer cell line, HT-29, that cancer cell motility is a crucial process that regulates histological growth pattern in CRCLM. HT29 CRC cells injected directly into the mouse liver grow into replacement HGPs, while HT29s silenced for ARPC3 grow into desmoplastic HGPs lesions. However, the molecular mechanisms that regulate ARPC3 in CRCLM remain unknown. To further dissect the molecular mechanisms differentiating desmoplastic from replacement HGPs, we performed RNA-seq analysis of CRCLM lesions from chemonaïve patients. Our data revealed that both TGFβ1 and RUNX1 were upregulated in RHGP comparing to DHGP lesions. This has been further validated by immunoblotting and immunohistochemistry. Consistently, RUNX1 has been reported as a downstream of TGFβ1 and transcriptional factor for ARPC3. Collectively, our data suggests that TGFβ1 and RUNX1 contribute to the formation of infiltrative type of colorectal cancer liver metastases possibly through upregulation of ARPC3. Note: This abstract was not presented at the meeting. Citation Format: Miran Rada, Anthoula Lazaris, Stephanie Petrillo, Abdellatif Amri, Peter Metrakos. Deciphering the molecular mechanisms driving infiltrative histopathological type of colorectal cancer liver metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5151.

Colorectal carcinoma (CRC) remains the third leading cause of cancer death in the Western world. Over 50% of CRC patients develop liver metastases (LM) and 90% will die from metastatic disease. Angiogenic inhibitors AIs (bevacizumab, Bev) were introduced with chemotherapy as first-line therapy for CRCLMs. While some patients respond well to this therapeutic strategy, the outcomes have fallen short of expectations. Recently we have reported the outcomes of resected CRCLM patients who have received neoadjuvant chemotherapy with or without AIs stratified by histologic growth patterns (HGPs): Desmoplastic HGP (DHGP), Pushing HGP (PHGP) and Replacement HGP (RHGP). The majority of metastases were either DHGPs, which derive the majority of their blood supply via sprouting angiogenesi,s or RHGP, which derive their blood supply from vessel co-option. When the neoadjuvant regimen included Bev, the DHGP patients more than doubled their 5-year overall survival (OS) compared to RHGP patients receiving the same Bev regimen. This OS difference was lost when both groups of patients were treated with chemo alone (no Bev). Although sprouting angiogenesis has been studied extensively and there is some understating of those mechanisms, we have no understanding of the mechanism behind co-option, and furthermore, apart from cytotoxic chemotherapy we have no targeted therapies for these patients. Theoretical Model: Cancer cells that arrive to the liver have to vascularize in order to increase in size, or as evidence has recently suggested, can grow avascularly in vascularized tissue by co-option of existing mature vessels. We hypothesize that there are several steps required for this process to occur. In our recent work, we have shown that cancer cell motility is essential for co-option. The angiopoietins-Tie2 mechanisms are critically involved in angiogenesis. Using human samples (chemonaïve, chemo-only and chemo plus Bev), we have evidence that Ang1-Tie2 mechanism is differently expressed in RHGP vs. DHGP metastases. The ratio of Ang2: Ang1 expression in DHGP tumors was higher compared to RHGP tumors, whereas VEGF appears to be equally expressed in both. Furthermore, a significant expression of Ang1 was detected in the hepatocytes at the interface region of the tumor and liver in RHGP. Thus, vascular quiescence maintained by Ang1-Tie2 mechanism prevails in RHGP verses DHGP metastasis; therefore this mechanism may be important in the development of vessel co-option vs. angiogenesis. Interestingly, Tie2 expression was expressed not only by vessels and tumor cells but also found in the leucocytes that were more abundant in DHGP vs. RHGP. This study will allow us to characterize the factors and mechanisms by which vessel co-option occurs in a metastatic setting and will stratify patients in terms of treatment. Citation Format: Nisreen Samir Ibrahim, Anthoula Lazaris, Stephanie Petrillo, Abdellatif Amri, Zu-Hua Gao, Peter Metrakos. Characterization of clinically relevant mechanism of resistance to angiogenic inhibitors in different growth patterns of human colorectal cancer liver metastases (CRCLM) by studying the angiopoietins-Tie2 mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5449.

In this paper I will consider the ethics of cloning as it occurs in Kazuo Ishiguro’s dystopian novel Never Let Me Go from the standpoint of a number of moral theories – consequentialism, natural law theory, Kantian moral theory, rights based theory, and virtue ethics. In light of the moral theories, I will develop an analysis for why cloning-for-biomedical-research as outlined in the 2002 document Human Cloning and Human Dignity by the President’s Council on Bioethics is morally permissible, while the cloning-based donation program in the novel is morally impermissible.

Colorectal cancer (CRC) is the third leading cause of cancer-related death in North America. Approximately 50% of patients will be diagnosed with CRC liver metastasis (CRLM) during the course of their disease. Untreated, patients will survive for only a few months, but with chemotherapy, a median survival of 20 months can be achieved. At present, no reliable indicators exist to predict outcome or prognosis in treatable patients. Moreover, no biological parameters are currently being considered for patient stratification into different treatment groups. We examined CRCLM resected from patients and have identified two major histologic growth patterns (HGP): a desmoplastic (DHGP) pattern and a replacement (RHGP) pattern where tumor cells replace parenchymal cells in the liver plates. These HGP involve distinct modes of angiogenesis and host cell responses. Namely, liver metastases with a RHGP grow by co-opting the stroma, without hypoxia-induced angiogenesis and with little perturbation of the liver architecture. In contrast, metastases with a DHGP show characteristics of ongoing, hypoxia-driven angiogenesis including increased fibrin deposition at the tumour-liver interface and increased endothelial cell proliferation. In addition RHGP is associated with poor clinical response to bevacizumab chemotherapy. These differences suggest that the two patterns trigger distinct microenvironment responses and as a consequence, may initiate and utilize different modes of vascularization and expansion. Our hypothesis is that there are distinct gene expression signatures in the tumor and/or host compartments of different HGPs, which will shed light on the biological mechanisms underlying this diversity of HGPs. To test this hypothesis we: i) have extracted high quality RNA from lesions of chemonaïve patients and through RNAseq analysis identified gene expression differences between DHGP, RHGP lesions ii.) to further understand the role of tumor associated vascularity, we have stained different lesions (chemonaïve, chemo only and chemo + bevacizumab) with vascular markers to look at tumor associated blood vessels (immature, intermediate and mature: aSMA1 & CD31) and the rate of endothelial cell proliferation (Ki67/CD34). Our preliminary data demonstrates the expression of neo-vessels in the DHGP desmoplastic ring and in the peripheral tumor along the parenchyma tissue have lower vascularity with fewer branches that are supplying the tumor. The RHGP has similar vasculature to the adjacent normal tissue with a sinusoidal network of blood supply and a higher vascularity than DHGP. We are now correlating these findings with RNAseq expression data. This work will result in the identification of targets in the HGPs that will help stratify patients in terms of treatment. We currently have less treatment options for the RHGP patients and it would be important to utilize the data from this study to develop new treatment strategies for those patients. Citation Format: Anthoula Lazaris, Abdellatif Amri, Pablo Zoroquiain, Stephanie K. Petrillo, Rafif Mattar, Zu-Hua Gao, Peter Vermeulen, Peter Metrakos. Vascularization of colorectal cancer liver metastasis: correlation with growth patterns. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1695.