Expression of the αvβ6 integrin is upregulated on multiple solid epithelial tumors while this integrin is largely absent on the healthy human epithelium. In the present study, we sought to compare the in vivo biodistributions of three αvβ6-selective agents that could be used for the positron emission tomography (PET)-based detection of malignancies expressing this integrin. A streptavidin-coupled αvβ6-binding peptide (streptavidin-A20), an anti-αvβ6 monoclonal antibody (620W.7) and an αvβ6-targeted protein, derived from a modified adenovirus 5 receptor-binding domain (Ad5.KO1.A20), were radiolabeled with [89Zr] and intravenously injected into mice bearing bilateral αvβ6-positive and -negative melanoma xenografts. Micro-PET imaging was performed at 0.33, 24, 48, 72, and (620W.7 group only) 144 h post-injection. Streptavidin-A20-based radiotracers preferentially accumulated in αvβ6-positive tumors, achieving >3.4:1 A375-β6:A375 activity ratios between 24 and 72 h post-injection, with off-target uptake restricted to the kidneys. 620W.7 exhibited roughly 2-fold greater accumulation in A375-β6 than A375 tumors, with lower levels of renal retention and greater activity detected in the liver and spleen. Despite having demonstrated selective binding to αvβ6-expressing cells in vitro, the Ad5.KO1.A20 protein failed to selectively traffic to tumors expressing this integrin in vivo. Nevertheless, streptavidin-A20 and 620W.7 demonstrated promise as αvβ6-selective agents for in vivo applications.

In homeostasis, counterbalanced morphogen signalling gradients along the vertical axis of the intestinal mucosa regulate the fate and function of epithelial and stromal cell compartments. Here, we use a disease-positioned mouse and human tissue to explore the consequences of pathological BMP signalling dysregulation on epithelial-mesenchymal interaction. Aberrant pan-epithelial expression of the secreted BMP antagonist Grem1 results in ectopic crypt formation, with lineage tracing demonstrating the presence of Lgr5(-) stem/progenitor cells. Isolated epithelial cell Grem1 expression has no effect on individual cell fate, indicating an intercompartmental impact of mucosal-wide BMP antagonism. Treatment with an anti-Grem1 antibody abrogates the polyposis phenotype, and triangulation of specific pathway inhibitors defines a pathological sequence of events, with Wnt-ligand-dependent ectopic stem cell niches forming through stromal remodelling following BMP disruption. These data support an emerging co-evolutionary model of intestinal cell compartmentalisation based on bidirectional regulation of epithelial-mesenchymal cell fate and function.

The Wnt signalling pathway plays a crucial role in tissue homeostasis and cancer biology due to its regulation of cellular processes, including proliferation, migration, and stem cell activity. Frizzled receptor 7 (FZD7) (a member of the F-class G protein-coupled receptors) has emerged as a key Wnt receptor within this pathway, which is elevated in several human malignancies. FZD7 is notably upregulated in gastrointestinal, breast, pancreatic, and hepatocellular carcinomas and transmits oncogenic Wnt signalling through canonical and non-canonical pathways. FZD7 promotes tumour initiation, and emerging evidence implicates FZD7 in cancer stem cell maintenance and epithelial–mesenchymal transition (EMT), reinforcing its role in metastasis. Therapeutic strategies targeting FZD7 have shown promise, including FZD7-specific monoclonal antibody-drug conjugates (ADCs), human single-chain fragment variable (scFVs) antibodies, and nanoparticles. Notably, our recent development of FZD7-ADC has demonstrated tumour-selective cytotoxicity with reduced off-target effects, positioning FZD7 as an attractive therapeutic target. Additionally, nanoparticle-based drug delivery systems have enhanced the precision of existing chemotherapies by targeting FZD7-expressing tumour cells. Despite significant advances, clinical translation remains a challenge due to potential on-target toxicity and the complexity of tumour microenvironments. Future research should focus on optimising delivery systems, refining antibody specificity, and conducting comprehensive preclinical and clinical trials. This review will focus on novel discoveries regarding FZD7 in cancer and provide an update on our original review on this subject in 2016. Additionally, we present new figures generated by our group using the publicly available Pan-Cancer Atlas RNAseq datasets, highlighting FZD7 expression patterns in patient samples. This integrated approach aims to provide updated insights into the function of FZD7 during cancer and its growing status as an attractive target for therapy. In summary, FZD7 stands out as a promising molecular target in cancer therapy due to its selective overexpression in tumours, functional role in Wnt-driven oncogenesis, and potential for innovative therapeutic applications. This review underscores the critical need for the continued exploration of FZD7-targeted therapies to improve patient outcomes in cancer treatment.

The intestinal epithelium has a remarkable capacity to regenerate after injury and DNA damage. Here, we show that the integrin effector protein Focal Adhesion Kinase (FAK) is dispensable for normal intestinal homeostasis and DNA damage signaling, but is essential for intestinal regeneration following DNA damage. Given Wnt/c-Myc signaling is activated following intestinal regeneration, we investigated the functional importance of FAK following deletion of the Apc tumor suppressor protein within the intestinal epithelium. Following Apc loss, FAK expression increased in a c-Myc-dependent manner. Codeletion of Apc and Fak strongly reduced proliferation normally induced following Apc loss, and this was associated with reduced levels of phospho-Akt and suppression of intestinal tumorigenesis in Apc heterozygous mice. Thus, FAK is required downstream of Wnt Signaling, for Akt/mTOR activation, intestinal regeneration, and tumorigenesis: Importantly, this work suggests that FAK inhibitors may suppress tumorigenesis in patients at high, risk of developing colorectal cancer.

In homeostasis, counterbalanced morphogen signalling gradients along the vertical axis of the intestinal mucosa regulate the fate and function of epithelial and stromal cell compartments. Here, we used a disease-positioned mouse, and human tissue, to explore the consequences of pathological Bone Morphogenetic Protein (BMP) signalling dysregulation on epithelial- mesenchymal interaction. Aberrant pan-epithelial expression of the secreted BMP antagonist GREM1, resulted in ectopic crypt formation with lineage tracing demonstrating the presence of Lgr5(-) stem/progenitor cells. Isolated epithelial cell Grem1 expression had no effect on individual cell fate, indicating an intercompartmental impact of mucosal-wide BMP antagonism. Treatment with a novel anti-Grem1 antibody abrogated the polyposis phenotype, and triangulation of specific pathway inhibitors defined a pathological sequence of events, with wnt-ligand dependent ectopic stem cell niches formed through stromal remodelling following BMP disruption. These data support an emerging co-evolutionary model of intestinal cell compartmentalisation based on bidirectional regulation of epithelial-mesenchymal cell fate and function. One Sentence Summary Pathological epithelial GREM1 expression induces therapeutically reversible ectopic stem cell niches through stromal remodelling ### Competing Interest Statement SJL has received grant income from UCB Pharma. GB was a UCB employee at the time the research was conducted. ND is an employee of UCB Pharma, UK and owns shares in UCB Pharma and Vertex Pharmaceuticals. All other authors have nothing to disclose.

As the treatment landscape for prostate cancer gradually evolves, the frequency of treatment-induced neuroendocrine prostate cancer (NEPC) and double-negative prostate cancer (DNPC) that is deficient for androgen receptor (AR) and neuroendocrine (NE) markers has increased. These prostate cancer subtypes are typically refractory to AR-directed therapies and exhibit poor clinical outcomes. Only a small range of NEPC/DNPC models exist, limiting our molecular understanding of this disease and hindering our ability to perform preclinical trials exploring novel therapies to treat NEPC/DNPC that are urgently needed in the clinic. Here, we report the development of the CU-PC01 PDX model that represents AR-negative mCRPC with PTEN/RB/PSMA loss and CTNN1B/TP53/BRCA2 genetic variants. The CU-PC01 model lacks classic NE markers, with only focal and/or weak expression of chromogranin A, INSM1 and CD56. Collectively, these findings are most consistent with a DNPC phenotype. Ex vivo and in vivo preclinical studies revealed that CU-PC01 PDX tumours are resistant to mCRPC standard-of-care treatments enzalutamide and docetaxel, mirroring the donor patient’s treatment response. Furthermore, short-term CU-PC01 tumour explant cultures indicate this model is initially sensitive to PARP inhibition with olaparib. Thus, the CU-PC01 PDX model provides a valuable opportunity to study AR-negative mCRPC biology and to discover new treatment avenues for this hard-to-treat disease.

Background: Pancreatic ductal adenocarcinoma (PDAC) represent an unmet clinical need. Approximately 90% of PDACs express high levels of alpha v beta 6 integrin. We have previously described Ad5(NULL)-A20, an adenovirus vector with ablated native means of cell entry and retargeted to alpha v beta 6 integrin by incorporation of an A20 peptide. Methods: Here, we incorporate suicide genes FCY1 and FCU1 encoding for cytosine deaminase (CDase) or a combination of CDase and UPRTase, capable of catalysing a non-toxic prodrug, 5-FC into the chemotherapeutic 5-FU and downstream metabolites, into replication-deficient Ad5 and Ad5(NULL)-A20. Results: We show that Ad5(NULL)-A20 enables the transfer of suicide genes to alpha v beta 6 integrin-positive PDAC cells which, in combination with 5-FC, results in cell death in vitro which is further mediated by a bystander effect in non-transduced cells. Intratumoural delivery of Ad5(NULL)-A20.FCU1 in combination with intraperitoneal delivery of 5-FC further results in tumour growth inhibition in a cell line xenograft in vivo. Using clinically-relevant 3D organoid models, we show selective transduction and therapeutic efficacy of FCU1 transgenes in combination with 5-FC. Conclusion: Taken together these data provide the preclinical rationale for combined Ad5(NULL)-A20.FCU1 plus 5-FC as a promising targeted therapy to mediate "in-tumour chemotherapy" and merits further investigation for the treatment of PDAC patients.

Colorectal cancer (CRC) is a complex disease with key oncogenic pathways, including Wnt, MAPK, and PI3K, co-operating to drive tumour initiation and progression. Loss-of-function mutations in the Wnt-pathway inhibitor APC are the most prominent genetic alterations and are commonly seen as the tumour-initiating event. Here, we have used genetically engineered mouse models to introduce pathway-activating mutations of PI3K (Pik3ca, Pten) and MAPK (Kras) signalling to the mouse gut. Comprehensive characterization of these models reveals pathway-dependent cooperation, leading to marked allele dose-dependent acceleration of tumour formation, underpinned by MYC-driven transcriptional reprogramming and alterations in downstream signalling pathways. We find the amino acid transporter SLC7A5 to be highly upregulated upon activation of PI3K signalling. In human CRCs, SLC7A5expression correlates with the newly defined PDS1 pathway-derived subtype and highly proliferative tumours. Genetic depletion of Slc7a5 in the newly developed PI3K-hyperactive models drastically extends survival by delaying tumour formation, even in aggressive Kras/Pik3caco-mutant mice. Finally, Slc7a5 gene deletion sensitizes these models to targeted MAPK inhibition. Taken together, SLC7A5 drives progression of PI3K-mutant CRCs and is an attractive (co-)target for mutation-specific inhibitors.

Figure S2. Gastric cancer cell xenografts treated with vehicle or OMP-18R5.

Figure S6. IHC for b-catenin, Myc, gastrin and Muc5a in Apc.Fzd7.TffCre adenoma tissue.

Supplementary Figure 6: Pik3ca+/HR and Ptenfl/fl prostate cancers acquire CRPC, while Pik3ca+/HR;Ptenfl/fl mutants are resistant to castration. (A) Representative H&E images of Pik3ca+/HR, Ptenfl/fl and Pik3ca+/HR;Ptenfl/fl anterior (AP) and ventral (VP) prostate lobes post-castration (scale bar: 50 um, n = 3). (B) Representative IHC images of Pik3ca+/HR, Ptenfl/fl and Pik3ca+/HR;Ptenfl/fl prostate tissue stained to detect Androgen receptor (AR) 2 weeks post-castration compared to uncastrated, age-matched controls (scale bar: 50 um, n = 3). Mice were castrated when prostate carcinoma was prevalent; Pik3ca+/HR = 400 d old, Ptenfl/fl = 200 d old and Pik3ca+/HR;Ptenfl/fl = 100 d old. Insert displays positive AR nuclei (arrows) in Pik3ca+/HR;Ptenfl/fl compound mutants (scale bar: 5 um). (C) Bar chart displaying total prostate weight normalised to body weight for Pik3ca+/HR mice 0, 2 and 42 weeks post-castration (n = 8, 7 and 7, respectively). Error bars: SEM, *P <0.05 compared to 0 weeks post-castration, or as indicated, one-way ANOVA with Tukey's multiple comparison test. (D) Representative H&E images of Pik3ca+/HR mice 0, 2 and 42 weeks post-castration (scale bar: 100 um). Mice were castrated at 100 d of age.

Supplemental Figure Legends. And Supp Table S1. List of Antibodies used. Table S2. Differential expression of genes in gp130 gastric adenomas compared to wild-type and following deletion of Fzd7.

Supplementary Figure 3 from K-ras and Wnt Signaling Synergize to Accelerate Prostate Tumorigenesis in the Mouse

Supplementary Figure 2 from K-ras and Wnt Signaling Synergize to Accelerate Prostate Tumorigenesis in the Mouse

Supplementary Tables 1-2 from PHLDA1 Expression Marks the Putative Epithelial Stem Cells and Contributes to Intestinal Tumorigenesis

Figure S7. Molecular and phenotypic characterisation of conditional deletion of Myc in Apc-mutant gastric adenomas.

Supplementary Figure 7: Characterization of Pik3ca+/HR, Ptenfl/fl and Pik3ca+/HR;Ptenfl/fl prostate tumors. Representative images of IHC to detect (A) PCNA and (B) Cleaved-caspase 3 (CC3) in Pik3ca+/HR, Ptenfl/fl and Pik3ca+/HR;Ptenfl/fl prostate tissue 2 weeks post-castration compared to uncastrated, age-matched controls (scale bar: 50 um, n = 3). Mice were castrated when prostate carcinoma was prevalent; Pik3ca+/HR = 400 d old, Ptenfl/fl = 200 d old and Pik3ca+/HR;Ptenfl/fl =100 d old. Quantitative RT-PCR to detect (C) Nkx3.1 and (D) Pbsn mRNA in Wt prostate and Pik3ca+/HR, Ptenfl/fl and Pik3ca+/HR;Ptenfl/fl stage-matched prostate carcinomas (n = 5). Error bars: SEM, *P <0.05 compared to Wt, or as indicated, one-way ANOVA with Tukey's multiple comparison test. (E) Western Blotting of protein lysates isolated from Wt prostate and stage-matched Pik3ca+/HR, Ptenfl/fl and Pik3ca+/HR;Ptenfl/fl prostate carcinomas to detect total AKT, p-AKT Thr308 and p-AKT Ser473 (n = 3).

BET protein expression in primary colorectal cancers.

Supplementary Figure 1 from K-ras and Wnt Signaling Synergize to Accelerate Prostate Tumorigenesis in the Mouse

Supplementary Figure 5: Pik3caH1047R mutation and Pten-deletion synergize to promote prostate cancer by increasing mTORC1/2 signaling. Histograms displaying phenotype incidence for anterior (A) and ventral (B) prostate lobes at 56 and 100 days of age. (C) Representative IHC images of Pik3ca+/HR;Ptenfl/fl prostate tumors at 100 d stained to detect CK8, CK5 and SMA (n = 3, scale bar: 50 um). (D) Bar chart displaying total prostate weight normalised to body weight for Wt (n = 7), Pik3ca+/HR (n = 8), Ptenfl/fl (n = 8) and Pik3ca+/HR;Ptenfl/fl (n = 7) 100 d old mice. Error bars: SEM, *P <0.05 compared to Wt or as indicated, one-way ANOVA with Tukey's multiple comparison test. (E) Quantitation of the apoptotic marker Cleaved-Caspase-3 (CC3) positive nuclei and (F) representative IHC images of CC3 staining in Pik3ca+/HR, Ptenfl/fl and Pik3ca+/HR;Ptenfl/fl stage-matched invasive prostate carcinoma (scale bar: 50 um, n = 3, one-way ANOVA with Tukey's multiple comparison test. Error bars: SEM). (G) Representative IHC images of Pik3ca+/HR, Ptenfl/fl and Pik3ca+/HR;Ptenfl/fl stage-matched invasive prostate carcinoma stained to detect p-AKT Thr308, p-RPS6 Ser235/236, p-4E-BP1 Thr37/46, p-AKT Ser473 and p-NDRG1 Thr346 (scale bar: 50 um). (H) Representative images of RNA in situ hybridisation (ISH) to detect positive (housekeeping gene PPIB, peptidylprolyl isomerase B) and negative (bacterial gene dapB) control probes to confirm RNA quality and the absence of background signal respectively (scale bar: 50 um, insert scale bar: 5 um).