Interplay between CCR7 and Notch1 axes promotes stemness in MMTV-PyMT mammary cancer cells

Background: Breast cancer is the major cause of cancer-related mortality in women. It is thought that quiescent stem-like cells within solid tumors are responsible for cancer maintenance, progression and eventual metastasis. We recently reported that the chemokine receptor CCR7, a multi-functional regulator of breast cancer, maintains the stem-like cell population. Methods: This study used a combination of molecular and cellular assays on primary mammary tumor cells from the MMTV-PyMT transgenic mouse with or without CCR7 to examine the signaling crosstalk between CCR7 and Notch pathways. Results: We show for the first time that CCR7 functionally intersects with the Notch signaling pathway to regulate mammary cancer stem-like cells. In this cell subpopulation, CCR7 stimulation activated the Notch signaling pathway, and deletion of CCR7 significantly reduced the levels of activated cleaved Notch1. Moreover, blocking Notch activity prevented specific ligand-induced signaling of CCR7 and augmentation of mammary cancer stem-like cell function. Conclusion: Crosstalk between CCR7 and Notch1 promotes stemness in mammary cancer cells and may ultimately potentiate mammary tumor progression. Therefore, dual targeting of both the CCR7 receptor and Notch1 signaling axes may be a potential therapeutic avenue to specifically inhibit the functions of breast cancer stem cells.Sarah T. Boyle, Krystyna A. Gieniec, Carly E. Gregor, Jessica W. Faulkner, Shaun R. McColl and Marina Kochetkov

The Origin and Contribution of Cancer-Associated Fibroblasts in Colorectal Carcinogenesis

Background & Aims: Cancer-associated fibroblasts (CAFs) play an important role in colorectal cancer (CRC) progression and predict poor prognosis in CRC patients. However, the cellular origins of CAFs remain unknown, making it challenging to therapeutically target these cells. Here, we aimed to identify the origins and contribution of colorectal CAFs associated with poor prognosis. Methods: To elucidate CAF origins, we used a colitis-associated CRC mouse model in 5 different fate-mapping mouse lines with 5-bromodeoxyuridine dosing. RNA sequencing of fluorescence-activated cell sorting–purified CRC CAFs was performed to identify a potential therapeutic target in CAFs. To examine the prognostic significance of the stromal target, CRC patient RNA sequencing data and tissue microarray were used. CRC organoids were injected into the colons of knockout mice to assess the mechanism by which the stromal gene contributes to colorectal tumorigenesis. Results: Our lineage-tracing studies revealed that in CRC, many ACTA2+ CAFs emerge through proliferation from intestinal pericryptal leptin receptor (Lepr)+ cells. These Lepr-lineage CAFs, in turn, express melanoma cell adhesion molecule (MCAM), a CRC stroma-specific marker that we identified with the use of RNA sequencing. High MCAM expression induced by transforming growth factor β was inversely associated with patient survival in human CRC. In mice, stromal Mcam knockout attenuated orthotopically injected colorectal tumoroid growth and improved survival through decreased tumor-associated macrophage recruitment. Mechanistically, fibroblast MCAM interacted with interleukin-1 receptor 1 to augment nuclear factor κB–IL34/CCL8 signaling that promotes macrophage chemotaxis. Conclusions: In colorectal carcinogenesis, pericryptal Lepr-lineage cells proliferate to generate MCAM+ CAFs that shape the tumor-promoting immune microenvironment. Preventing the expansion/differentiation of Lepr-lineage CAFs or inhibiting MCAM activity could be effective therapeutic approaches for CRC

Angular Distributions of Secondary Electrons Emitted from the Polycrystalline Silver

The angular distributions of secondary electrons (including Auger electrons) of different energies emitted from polycrystalline silver bombarded with primary electrons (E$\text{}_{p}$ = 1000 and 2500 eV) were measured with a RFA analyzer equipped with an additional collector (acceptance angle ≈ 4°). Additionally, the angular distributions of primary electrons of different energies elastically backscattered from the same sample were also investigated by means of a specially constructed rotable RFA analyzer equipped with a channeltron. The results obtained show the cosine distribution of secondary and Auger electrons emitted as shown previously in the literature. The angular distributions of electrons backscattered elastically depend on electron energy and are in good agreement with theoretical calculations (Monte Carlo simulation of primary electrons trajectories in solids) proposed by Jabłoński

Genetic editing of colonic organoids provides a molecularly distinct and orthotopic preclinical model of serrated carcinogenesis

Serrated colorectal cancer (CRC) accounts for approximately 25% of cases and includes tumours that are among the most treatment resistant and with worst outcomes. This CRC subtype is associated with activating mutations in the mitogen-activated kinase pathway gene, BRAF, and epigenetic modifications termed the CpG Island Methylator Phenotype, leading to epigenetic silencing of key tumour suppressor genes. It is still not clear which (epi-)genetic changes are most important in neoplastic progression and we begin to address this knowledge gap herein.We use organoid culture combined with CRISPR/Cas9 genome engineering to sequentially introduce genetic alterations associated with serrated CRC and which regulate the stem cell niche, senescence and DNA mismatch repair.Targeted biallelic gene alterations were verified by DNA sequencing. Organoid growth in the absence of niche factors was assessed, as well as analysis of downstream molecular pathway activity. Orthotopic engraftment of complex organoid lines, but not BrafV600E alone, quickly generated adenocarcinoma in vivo with serrated features consistent with human disease. Loss of the essential DNA mismatch repair enzyme, Mlh1, led to microsatellite instability. Sphingolipid metabolism genes are differentially regulated in both our mouse models of serrated CRC and human CRC, with key members of this pathway having prognostic significance in the human setting.We generate rapid, complex models of serrated CRC to determine the contribution of specific genetic alterations to carcinogenesis. Analysis of our models alongside patient data has led to the identification of a potential susceptibility for this tumour type.Tamsin R M Lannagan, Young K Lee, Tontong Wang ... Laura Vrbanac ... Roshini Somashekar, Krystyna Gieniec, Miao Yang, Jia Q Ng, Nobumi Suzuki, Mari Ichinose, Josephine A Wright, Hiroki Kobayashi ... Daniel L Worthley, Susan L. Wood

Portal Vein Injection of Colorectal Cancer Organoids to Study the Liver Metastasis Stroma

Hepatic metastasis of colorectal cancer (CRC) is a leading cause of cancer-related death. Cancer-associated fibroblasts (CAFs), a major component of the tumor microenvironment, play a crucial role in metastatic CRC progression and predict poor patient prognosis. However, there is a lack of satisfactory mouse models to study the crosstalk between metastatic cancer cells and CAFs. Here, we present a method to investigate how liver metastasis progression is regulated by the metastatic niche and possibly could be restrained by stroma-directed therapy. Portal vein injection of CRC organoids generated a desmoplastic reaction, which faithfully recapitulated the fibroblast-rich histology of human CRC liver metastases. This model was tissue-specific with a higher tumor burden in the liver when compared to an intra-splenic injection model, simplifying mouse survival analyses. By injecting luciferase-expressing tumor organoids, tumor growth kinetics could be monitored by in vivo imaging. Moreover, this preclinical model provides a useful platform to assess the efficacy of therapeutics targeting the tumor mesenchyme. We describe methods to examine whether adeno-associated virus-mediated delivery of a tumor-inhibiting stromal gene to hepatocytes could remodel the tumor microenvironment and improve mouse survival. This approach enables the development and assessment of novel therapeutic strategies to inhibit hepatic metastasis of CRC.Hiroki Kobayashi, Krystyna A. Gieniec, Jia Q. Ng, Jarrad Goyne, Tamsin R. M. Lannagan, Elaine M. Thomas, Georgette Radford, Tongtong Wang, Nobumi Suzuki, Mari Ichinose, Josephine A. Wright, Laura Vrbanac, Alastair D. Burt, Masahide Takahashi, Atsushi Enomoto, Daniel L. Worthley, Susan L. Wood

Stromal DLK1 promotes proliferation and inhibits differentiation of the intestinal epithelium during development

First published January 20, 2021BACKGROUND & AIMS: The stem/progenitor cells of the developing intestine arebiologically distinct from their adult counterparts. Here we examine the microenvironmental cues that regulate the embryonic stem/progenitor population, focusing on the role of Notch pathway factor, Delta-Like Protein 1 (DLK1). METHODS: mRNAseq analyses of intestinal mesenchymal cells (IMC) collected from embryonic day 14.5 (E14.5) or adult IMCs and a novel co-culture system with E14.5 intestinal epithelial organoids were used. Following addition of recombinant DLK1 (rDLK) or Dlk1 siRNA (siDlk1), epithelial characteristics were compared using imaging, replating efficiency assays, qPCR and immunocytochemistry. The intestinal phenotype of littermate Dlk1 +/+ and Dlk1 -/- mice was compared using immunohistochemistry. RESULTS: Using transcriptomic analyses we identified morphogens derived from the embryonic mesenchyme that potentially regulate the developing epithelial cells, to focus on Notch family candidate, DLK1. Immunohistochemistry indicated that DLK1 was expressed exclusively in the intestinal stroma at E14.5 at the top of emerging villi, decreased after birth and shifted to the intestinal epithelium in adulthood. In co-culture experiments, addition of rDLK1 to adult IMCs inhibited organoid differentiation, whereas Dlk1 knock-down in embryonic IMCs increased epithelial differentiation to secretory lineage cells. Dlk1 -/- mice had restricted Ki67+ cells in the villi base and increased secretory lineage cells compared with Dlk1 +/+ embryos. CONCLUSIONS: Mesenchyme-derived DLK1 plays an important role in the promotion of epithelial stem/precursor expansion and prevention of differentiation to secretory lineages in the developing intestine.Mari Ichinose, Nobumi Suzuki, Tongtong Wang, Josephine A. Wright, Tamsin R. M. Lannagan, Laura Vrbanac ... et al

The origin and contribution of cancer-associated fibroblasts in colorectal carcinogenesis

BACKGROUND AND AIMS: Cancer-associated fibroblasts (CAFs) play an important role in colorectal cancer (CRC) progression and predict poor prognosis in CRC patients. However, the cellular origins of CAFs remain unknown, making it challenging to therapeutically target these cells. Here, we aimed to identify the origins and contribution of colorectal CAFs associated with poor prognosis. METHODS: To elucidate CAF origins, we used a colitis-associated CRC mouse model in 5 different fate-mapping mouse lines with 5- bromodeoxyuridine dosing. RNA sequencing of fluorescence-activated cell sorting–purified CRC CAFs was performed to identify a potential therapeutic target in CAFs. To examine the prognostic significance of the stromal target, CRC patient RNA sequencing data and tissue microarray were used. CRC organoids were injected into the colons of knockout mice to assess the mechanism by which the stromal gene contributes to colorectal tumorigenesis. RESULTS: Our lineage-tracing studies revealed that in CRC, many ACTA2þ CAFs emerge through proliferation from intestinal pericryptal leptin receptor (Lepr) þ cells. These Lepr-lineage CAFs, in turn, express melanoma cell adhesion molecule (MCAM), a CRC stroma-specific marker that we identified with the use of RNA sequencing. High MCAM expression induced by transforming growth factor b was inversely associated with patient survival in human CRC. In mice, stromal Mcam knockout attenuated orthotopically injected colorectal tumoroid growth and improved survival through decreased tumor-associated macrophage recruitment. Mechanistically, fibroblast MCAM interacted with interleukin-1 receptor 1 to augment nuclear factor kB–IL34/CCL8 signalling that promotes macrophage chemotaxis. CONCLUSIONS: In colorectal carcinogenesis, pericryptal Lepr-lineage cells proliferate to generate MCAMþ CAFs that shape the tumor-promoting immune microenvironment. Preventing the expansion/differentiation of Leprlineage CAFs or inhibiting MCAM activity could be effective therapeutic approaches for CRC.Hiroki Kobayashi ... Alastair D. Burt ... Lisa M. Butler ... Georgette A. Radford ... Tarik Sammour ... Susan L. Woods ... et al

The Antianginal Drug Perhexiline Displays Cytotoxicity against Colorectal Cancer Cells In Vitro: A Potential for Drug Repurposing

Colorectal cancer (CRC) is the second leading cause of cancer-related death worldwide. Perhexiline, a prophylactic anti-anginal drug, has been reported to have anti-tumour effects both in vitro and in vivo. Perhexiline as used clinically is a 50:50 racemic mixture ((R)-P) of (−) and (+) enantiomers. It is not known if the enantiomers differ in terms of their effects on cancer. In this study, we examined the cytotoxic capacity of perhexiline and its enantiomers ((−)-P and (+)-P) on CRC cell lines, grown as monolayers or spheroids, and patient-derived organoids. Treatment of CRC cell lines with (R)-P, (−)-P or (+)-P reduced cell viability, with IC50 values of ~4 µM. Treatment was associated with an increase in annexin V staining and caspase 3/7 activation, indicating apoptosis induction. Caspase 3/7 activation and loss of structural integrity were also observed in CRC cell lines grown as spheroids. Drug treatment at clinically relevant concentrations significantly reduced the viability of patient-derived CRC organoids. Given these in vitro findings, perhexiline, as a racemic mixture or its enantiomers, warrants further investigation as a repurposed drug for use in the management of CRC