Orthotopic MC-38 Allograft as a Robust Preclinical Model of Colorectal Carcinoma.

Colorectal cancer (CRC) is a significant global health concern, requiring effective preclinical models for studying its development and testing therapies. Mouse models have been used, including spontaneous tumors, carcinogen exposure, and tumor cell implantation as xenografts or at orthotopic sites. Here, we describe an orthotopic preclinical model of CRC, which provides a valuable tool for studying tumor growth and the tumor microenvironment, offering a more accurate representation of human CRC compared to xenograft models.

Combination of bazedoxifene with chemotherapy and SMAC-mimetics for the treatment of colorectal cancer.

Excessive STAT3 signalling via gp130, the shared receptor subunit for IL-6 and IL-11, contributes to disease progression and poor survival outcomes in patients with colorectal cancer. Here, we provide evidence that bazedoxifene inhibits tumour growth via direct interaction with the gp130 receptor to suppress IL-6 and IL-11-mediated STAT3 signalling. Additionally, bazedoxifene combined with chemotherapy synergistically reduced cell proliferation and induced apoptosis in patient-derived colon cancer organoids. We elucidated that the primary mechanism of anti-tumour activity conferred by bazedoxifene treatment occurs via pro-apoptotic responses in tumour cells. Co-treatment with bazedoxifene and the SMAC-mimetics, LCL161 or Birinapant, that target the IAP family of proteins, demonstrated increased apoptosis and reduced proliferation in colorectal cancer cells. Our findings provide evidence that bazedoxifene treatment could be combined with SMAC-mimetics and chemotherapy to enhance tumour cell apoptosis in colorectal cancer, where gp130 receptor signalling promotes tumour growth and progression.

Screening for Primary Aldosteronism by Mass Spectrometry Versus Immunoassay Measurements of Aldosterone: A Prospective Within-Patient Study.

BACKGROUND: Measurements of aldosterone by mass spectrometry are more accurate and less prone to interferences than immunoassay measurements, and may produce a more accurate aldosterone:renin ratio (ARR) when screening for primary aldosteronism (PA). METHODS: Differences in diagnostic performance of the ARR using mass spectrometry vs immunoassay measurements of aldosterone were examined in 710 patients screened for PA. PA was confirmed in 153 patients and excluded in 451 others. Disease classifications were not achieved in 106 patients. Areas under receiver-operating characteristic curves (AUROC) and other measures were used to compare diagnostic performance. RESULTS: Mass spectrometry-based measurements yielded lower plasma aldosterone concentrations than immunoassay measurements. For the ARR based on immunoassay measurements of aldosterone, AUROCs were slightly lower (P = 0.018) than those using mass spectrometry measurements (0.895 vs 0.906). The cutoff for the ARR to reach a sensitivity of 95% was 30 and 21.5 pmol/mU by respective immunoassay and mass spectrometry-based measurements, which corresponded to specificities of 57% for both. With data restricted to patients with unilateral PA, diagnostic sensitivities of 94% with specificities >81% could be achieved at cutoffs of 68 and 52 pmol/mU for respective immunoassay and mass spectrometry measurements. CONCLUSIONS: Mass spectrometry-based measurements of aldosterone for the ARR provide no clear diagnostic advantage over immunoassay-based measurements. Both approaches offer limited diagnostic accuracy for the ARR as a screening test. One solution is to employ the higher cutoffs to triage patients likely to have unilateral PA for further tests and possible adrenalectomy, while using the lower cutoffs to identify others for targeted medical therapy.German Clinical Trials Register ID: DRKS00017084.

Identification of Serum Biomarkers to Monitor Therapeutic Response in Intestinal-Type Gastric Cancer.

There are a limited number of clinically useful serum biomarkers to predict tumor onset or treatment response in gastric cancer (GC). For this reason, we explored the serum proteome of the gp130Y757F murine model of intestinal-type gastric cancer (IGC). We identified 30 proteins with significantly elevated expression in early gp130Y757F IGC and 12 proteins that were significantly elevated in late gp130Y757F IGC compared to age- and gender-matched wild-type mice. Within these signatures, there was an overlap of 10 proteins commonly elevated in both early- and late-stage disease. These results highlight the potential to identify serum biomarkers of disease stage. Since IGC in the gp130Y757F model can be reversed following therapeutic inhibition of Interleukin (IL)-11, we explored whether the protein signatures we identified could be used to monitor tumor regression. We compared two different therapeutic modalities and found 5 proteins to be uniquely differentially expressed between control animals and animals halfway through treatment, with 10 differentially expressed at the end of treatment. Our findings highlight the potential to identify reliable biomarkers to track IGC tumor regression in response to treatment.

Mechanisms of cellular crosstalk in the gastric tumor microenvironment are mediated by YAP1 and STAT3.

Deregulation of the Hippo pathway is a driver for cancer progression and treatment resistance. In the context of gastric cancer, YAP1 is a biomarker for poor patient prognosis. Although genomic tumor profiling provides information of Hippo pathway activation, the present study demonstrates that inhibition of Yap1 activity has anti-tumor effects in gastric tumors driven by oncogenic mutations and inflammatory cytokines. We show that Yap1 is a key regulator of cell metabolism, proliferation, and immune responses in normal and neoplastic gastric epithelium. We propose that the Hippo pathway is targetable across gastric cancer subtypes and its therapeutic benefits are likely to be mediated by both cancer cell-intrinsic and -extrinsic mechanisms.

Tumor macrophage functional heterogeneity can inform the development of novel cancer therapies.

Macrophages represent a key component of the tumor microenvironment (TME) and are largely associated with poor prognosis. Therapeutic targeting of macrophages has historically focused on inhibiting their recruitment or reprogramming their phenotype from a protumor (M2-like) to an antitumor (M1-like) one. Unfortunately, this approach has not provided clinical breakthroughs that have changed practice. Emerging studies utilizing single-cell RNA-sequencing (scRNA-seq) and spatial transcriptomics have improved our understanding of the ontogeny, phenotype, and functional plasticity of macrophages. Overlaying the wealth of current information regarding macrophage molecular subtypes and functions has also identified novel therapeutic vulnerabilities that might drive better control of tumor-associated macrophages (TAMs). Here, we discuss the functional profiling of macrophages and provide an update of novel macrophage-targeted therapies in development.

A tuft cell - ILC2 signaling circuit provides therapeutic targets to inhibit gastric metaplasia and tumor development.

Although gastric cancer is a leading cause of cancer-related deaths, systemic treatment strategies remain scarce. Here, we report the pro-tumorigenic properties of the crosstalk between intestinal tuft cells and type 2 innate lymphoid cells (ILC2) that is evolutionarily optimized for epithelial remodeling in response to helminth infection. We demonstrate that tuft cell-derived interleukin 25 (IL25) drives ILC2 activation, inducing the release of IL13 and promoting epithelial tuft cell hyperplasia. While the resulting tuft cell - ILC2 feed-forward circuit promotes gastric metaplasia and tumor formation, genetic depletion of tuft cells or ILC2s, or therapeutic targeting of IL13 or IL25 alleviates these pathologies in mice. In gastric cancer patients, tuft cell and ILC2 gene signatures predict worsening survival in intestinal-type gastric cancer where ~40% of the corresponding cancers show enriched co-existence of tuft cells and ILC2s. Our findings suggest a role for ILC2 and tuft cells, along with their associated cytokine IL13 and IL25 as gatekeepers and enablers of metaplastic transformation and gastric tumorigenesis, thereby providing an opportunity to therapeutically inhibit early-stage gastric cancer through repurposing antibody-mediated therapies.

TCF-1 limits intraepithelial lymphocyte antitumor immunity in colorectal carcinoma.

Intraepithelial lymphocytes (IELs), including αß and γδ T cells (T-IELs), constantly survey and play a critical role in maintaining the gastrointestinal epithelium. We show that cytotoxic molecules important for defense against cancer were highly expressed by T-IELs in the small intestine. In contrast, abundance of colonic T-IELs was dependent on the microbiome and displayed higher expression of TCF-1/TCF7 and a reduced effector and cytotoxic profile, including low expression of granzymes. Targeted deletion of TCF-1 in γδ T-IELs induced a distinct effector profile and reduced colon tumor formation in mice. In addition, TCF-1 expression was significantly reduced in γδ T-IELs present in human colorectal cancers (CRCs) compared with normal healthy colon, which strongly correlated with an enhanced γδ T-IEL effector phenotype and improved patient survival. Our work identifies TCF-1 as a colon-specific T-IEL transcriptional regulator that could inform new immunotherapy strategies to treat CRC.

A Mouse Model for the Rapid and Binomial Assessment of Putative WNT/ß-Catenin Signalling Inhibitors.

Specific signalling thresholds of the WNT/ß-catenin pathway affect embryogenesis and tissue homeostasis in the adult, with mutations in this pathway frequently occurring in cancer. Excessive WNT/ß-catenin activity inhibits murine anterior development associated with embryonic lethality and accounts for the driver event in 80% of human colorectal cancers. Uncontrolled WNT/ß-catenin signalling arises primarily from impairment mutation in the tumour suppressor gene APC that otherwise prevents prolonged stabilisation of ß-catenin. Surprisingly, no inhibitor compounds for WNT/ß-catenin signalling have reached clinical use in part owing to the lack of specific in vivo assays that discriminate between on-target activities and dose-limiting toxicities. Here, we present a simple in vivo assay with a binary outcome whereby the administration of candidate compounds to pregnant and phenotypically normal Apcflox/flox mice can rescue in utero death of Apcmin/flox mutant conceptus without subsequent post-mortem assessment of WNT/ß-catenin signalling. Indeed, the phenotypic plasticity of born Apcmin/flox conceptus enables future refinement of our assay to potentially enable dosage finding and cross-compound comparisons. Thus, we show for the first time the suitability of endogenous WNT/ß-catenin signalling during embryonic development to provide an unambiguous and sensitive mammalian in vivo model to assess the efficacy and bioavailability of potential WNT/ß-catenin antagonists.

Functional roles of SRC signaling in pancreatic cancer: Recent insights provide novel therapeutic opportunities.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignant disease with a 5-year survival rate of <10%. Aberrant activation or elevated expression of the tyrosine kinase c-SRC (SRC) is frequently observed in PDAC and is associated with a poor prognosis. Preclinical studies have revealed a multifaceted role for SRC activation in PDAC, including promoting chronic inflammation, tumor cell proliferation and survival, cancer cell stemness, desmoplasia, hypoxia, angiogenesis, invasion, metastasis, and drug resistance. Strategies to inhibit SRC signaling include suppressing its catalytic activity, inhibiting protein stability, or by interfering with signaling components of the SRC signaling pathway including suppressing protein interactions of SRC. In this review, we discuss the molecular and immunological mechanisms by which aberrant SRC activity promotes PDAC tumorigenesis. We also provide a comprehensive update of SRC inhibitors in the clinic, and discuss the clinical challenges associated with targeting SRC in pancreatic cancer.

Structure-Guided Prediction of the Functional Impact of DCLK1 Mutations on Tumorigenesis.

Doublecortin-like kinase 1 (DCLK1) is a functional serine/threonine (S/T)-kinase and a member of the doublecortin family of proteins which are characterized by their ability to bind to microtubules (MTs). DCLK1 is a proposed cancer driver gene, and its upregulation is associated with poor overall survival in several solid cancer types. However, how DCLK1 associates with MTs and how its kinase function contributes to pro-tumorigenic processes is poorly understood. This review builds on structural models to propose not only the specific functions of the domains but also attempts to predict the impact of individual somatic missense mutations on DCLK1 functions. Somatic missense mutations in DCLK1 are most frequently located within the N-terminal MT binding region and likely impact on the ability of DCLK1 to bind to αß-tubulin and to polymerize and stabilize MTs. Moreover, the MT binding affinity of DCLK1 is negatively regulated by its auto-phosphorylation, and therefore mutations that affect kinase activity are predicted to indirectly alter MT dynamics. The emerging picture portrays DCLK1 as an MT-associated protein whose interactions with tubulin heterodimers and MTs are tightly controlled processes which, when disrupted, may confer pro-tumorigenic properties.

Longitudinal quantification of mouse gastric tumor organoid viability and growth using luminescence and microscopy.

Tumor-derived organoids are valuable for testing anti-cancer drugs in vitro, but existing lysis-based protocols for viability measurement are laborious and restricted at a single time point. Here, we provide a lysis-free protocol for longitudinal and rapid assessment of mouse gastric tumor organoid viability and growth. We describe organoid plating, viability assessment via luminescence measurement, quantification of organoid growth by microscopy imaging, and treatment of organoids with test compounds to evaluate the effects on viability and growth at various time points.

Generation of gene-of-interest knockouts in murine organoids using CRISPR-Cas9.

Gene-of-interest knockout organoids present a powerful and versatile research tool to study a gene's effects on many biological and pathological processes. Here, we present a straightforward and broadly applicable protocol to generate gene knockouts in mouse organoids using CRISPR-Cas9 technology. We describe the processes of transient transfecting organoids with pre-assembled CRISPR-Cas9 ribonucleoprotein complexes, organoid cell sorting, and establishing clonal organoid culture pairs. We then detail how to confirm the knockout via Western blot analysis.

An intrasplenic injection model of pancreatic cancer metastasis to the liver in mice.

Here, we provide a protocol for an intrasplenic injection model to establish pancreatic tumors in the mouse liver. We describe the steps to inject tumor cells into mouse spleen and to perform a splenectomy, followed by animal recovery and end point analysis of tumors in the liver. This model allows rapid and reproducible tumor growth in a clinically relevant metastatic site, providing a platform to evaluate the efficacy of anti-cancer drugs. This technique can be expanded to other cancer cell lines. For complete details on the use and execution of this protocol, please refer to Poh et al. (2022).1.

Protocol for investigating tertiary lymphoid structures in human and murine fixed tissue sections using Opal™-TSA multiplex immunohistochemistry.

Tertiary lymphoid structures (TLSs) are ectopic lymphoid aggregates that can develop within or adjacent to tumors, but protocols that can accurately identify and characterize TLSs are lacking. Here, we present a protocol for the in situ interrogation and characterization of TLSs in human and murine tissue sections using Opal™-tyramide signal amplification multiplex immunohistochemistry. This protocol enables simultaneous detection of up to 7 markers (6 antigens and a DAPI counterstain). We also describe a grading system to identify immature and mature TLSs.

Improved glucose tolerance after initiation of Elexacaftor / Tezacaftor / Ivacaftor in adults with cystic fibrosis.

BACKGROUND: The novel triple CFTR modulator therapy Elexacaftor/Tezacaftor/Ivacaftor (ELX/TEZ/IVA) improves lung function, body mass index (BMI), sinus clearance, and quality of life in patients with cystic fibrosis. Whether treatment with ELX/TEZ/IVA is associated with improved glucose tolerance is unknown. METHODS: This cohort study included adults with CF and at least one copy of F508del.. Study assessments before treatment and at least 3 months after ELX/TEZ/IVA initiation included an oral glucose tolerance test (OGTT) with glucose and insulin measurements, BMI, lung function test, and sweat chloride levels. We used an analysis of response profiles to calculate changes in outcomes. RESULTS: 33 patients (27.8 ± 6.3 years; 73% male; 64% F508del homozygous) were included. After a median of 184 [IQR, 107 - 278] days following treatment initiation 16 (48.5%) patients improved their glucose tolerance category, while 13 (39.4%) remained unchanged and 4 (12.1%) deteriorated. Overall, 60, 90 and 120 min OGTT glycemia decreased significantly from 11.9 ± 2.7 mmol/l to 10.6 ± 2.8 mmol/l (p = 0.012), 10.4 ± 3.0 mmol/l to 8.4 ± 3.6 mmol/l (p = 0.002) and 7.3 ± 3.1 mmol/l to 5.7 ± 3.0 mmol/l (p = 0.012). HbA1c levels also improved significantly, from 5.50±0.24% to 5.39±0.25% (p = 0.039). CONCLUSION: In adult patients with CF and at least one copy of F508del, treatment with the triple CFTR modulator was associated with possible improvement of glucose tolerance without increases of insulin secretion. Early initiation of treatment as assessed through long-term prospective trials is mandatory to demonstrate if decreased glucose control is preventable or even reversible.

Genotype-Tailored ERK/MAPK Pathway and HDAC Inhibition Rewires the Apoptotic Rheostat to Trigger Colorectal Cancer Cell Death.

The EGFR/RAS/MEK/ERK signaling pathway (ERK/MAPK) is hyperactivated in most colorectal cancers. A current limitation of inhibitors of this pathway is that they primarily induce cytostatic effects in colorectal cancer cells. Nevertheless, these drugs do induce expression of proapoptotic factors, suggesting they may prime colorectal cancer cells to undergo apoptosis. As histone deacetylase inhibitors (HDACis) induce expression of multiple proapoptotic proteins, we examined whether they could synergize with ERK/MAPK inhibitors to trigger colorectal cancer cell apoptosis. Combined MEK/ERK and HDAC inhibition synergistically induced apoptosis in colorectal cancer cell lines and patient-derived tumor organoids in vitro, and attenuated Apc-initiated adenoma formation in vivo. Mechanistically, combined MAPK/HDAC inhibition enhanced expression of the BH3-only proapoptotic proteins BIM and BMF, and their knockdown significantly attenuated MAPK/HDAC inhibitor-induced apoptosis. Importantly, we demonstrate that the paradigm of combined MAPK/HDAC inhibitor treatment to induce apoptosis can be tailored to specific MAPK genotypes in colorectal cancers, by combining an HDAC inhibitor with either an EGFR, KRASG12C or BRAFV600 inhibitor in KRAS/BRAFWT; KRASG12C, BRAFV600E colorectal cancer cell lines, respectively. These findings identify a series of ERK/MAPK genotype-tailored treatment strategies that can readily undergo clinical testing for the treatment of colorectal cancer.

Tumor Growth Remains Refractory to Myc Ablation in Host Macrophages.

Aberrant expression of the oncoprotein c-Myc (Myc) is frequently observed in solid tumors and is associated with reduced overall survival. In addition to well-recognized cancer cell-intrinsic roles of Myc, studies have also suggested tumor-promoting roles for Myc in cells of the tumor microenvironment, including macrophages and other myeloid cells. Here, we benchmark Myc inactivation in tumor cells against the contribution of its expression in myeloid cells of murine hosts that harbor endogenous or allograft tumors. Surprisingly, we observe that LysMCre-mediated Myc ablation in host macrophages does not attenuate tumor growth regardless of immunogenicity, the cellular origin of the tumor, the site it develops, or the stage along the tumor progression cascade. Likewise, we find no evidence for Myc ablation to revert or antagonize the polarization of alternatively activated immunosuppressive macrophages. Thus, we surmise that systemic targeting of Myc activity may confer therapeutic benefits primarily through limiting Myc activity in tumor cells rather than reinvigorating the anti-tumor activity of macrophages.

Inhibition of HCK in myeloid cells restricts pancreatic tumor growth and metastasis.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with a low 5-year survival rate and is associated with poor response to therapy. Elevated expression of the myeloid-specific hematopoietic cell kinase (HCK) is observed in PDAC and correlates with reduced patient survival. To determine whether aberrant HCK signaling in myeloid cells is involved in PDAC growth and metastasis, we established orthotopic and intrasplenic PDAC tumors in wild-type and HCK knockout mice. Genetic ablation of HCK impaired PDAC growth and metastasis by inducing an immune-stimulatory endotype in myeloid cells, which in turn reduced the desmoplastic microenvironment and enhanced cytotoxic effector cell infiltration. Consequently, genetic ablation or therapeutic inhibition of HCK minimized metastatic spread, enhanced the efficacy of chemotherapy, and overcame resistance to anti-PD1, anti-CTLA4, or stimulatory anti-CD40 immunotherapy. Our results provide strong rationale for HCK to be developed as a therapeutic target to improve the response of PDAC to chemo- and immunotherapy.

Crosstalk between epithelium, myeloid and innate lymphoid cells during gut homeostasis and disease.

The gut epithelium not only provides a physical barrier to separate a noxious outside from a sterile inside but also allows for highly regulated interactions between bacteria and their products, and components of the immune system. Homeostatic maintenance of an intact epithelial barrier is paramount to health, requiring an intricately regulated and highly adaptive response of various cells of the immune system. Prolonged homeostatic imbalance can result in chronic inflammation, tumorigenesis and inefficient antitumor immune control. Here we provide an update on the role of innate lymphoid cells, macrophages and dendritic cells, which collectively play a critical role in epithelial barrier maintenance and provide an important linkage between the classical innate and adaptive arm of the immune system. These interactions modify the capacity of the gut epithelium to undergo continuous renewal, safeguard against tumor formation and provide feedback to the gut microbiome, which acts as a seminal contributor to cellular homeostasis of the gut.