A high-fat diet can lead to gut microbiota dysbiosis, chronic intestinal inflammation, and metabolic syndrome. Notably, resulting phenotypes, such as glucose and insulin levels, colonic crypt cell proliferation, and macrophage infiltration, exhibit sex differences, and females are less affected. This is, in part, attributed to sex hormones. To investigate if there are sex differences in the microbiota and if estrogenic ligands can attenuate high-fat diet-induced dysbiosis, we used whole-genome shotgun sequencing to characterize the impact of diet, sex, and estrogenic ligands on the microbial composition of the cecal content of mice. We here report clear host sex differences along with remarkably sex-dependent responses to high-fat diet. Females, specifically, exhibited increased abundance of Blautia hansenii, and its levels correlated negatively with insulin levels in both sexes. Estrogen treatment had a modest impact on the microbiota diversity but altered a few important species in males. This included Collinsella aerofaciens F, which we show correlated with colonic macrophage infiltration. In conclusion, male and female mice exhibit clear differences in their cecal microbial composition and in how diet and estrogens impact the composition. Further, specific microbial strains are significantly correlated with metabolic parameters.

There are significant sex differences in colorectal cancer (CRC), including in incidence, onset, and molecular characteristics. Further, while inflammatory bowel disease (IBD) is a risk factor for CRC in both sexes, men with IBD have a 60% higher risk of developing CRC compared to women. In this study, we investigated sex differences during colitis-associated CRC (CAC) using a chemically induced CAC mouse model. The mice were treated with azoxymethane (AOM) and dextran sodium sulfate (DSS) and followed for 9 and 15 weeks. We performed RNA-sequencing of colon samples from males (n = 15) and females (n = 15) to study different stages of inflammation and identify corresponding transcriptomic sex differences in non-tumor colon tissue. We found a significant transcriptome response to AOM/DSS treatment in both sexes, including in pathways related to inflammation and cell proliferation. Notably, we found a stronger response in males and that male-specific differentially expressed genes were involved in NFκB signaling and circadian rhythm. Further, an overrepresented proportion of male-specific gene regulations were predicted to be targets of Stat3, whereas for females, targets of the glucocorticoid receptor (Gr/Nr3c1) were overrepresented. At 15 weeks, the most apparent sex difference involved genes with functions in T cell proliferation, followed by the regulation of demethylases. The majority of sex differences were thus related to inflammation and the immune system. Our novel data, profiling the transcriptomic response to chemically induced colitis and CAC, indicate clear sex differences in CRC initiation and progression.

Human papillomavirus (HPV)‐associated tonsillar squamous cell carcinoma (TSCC) is suggested to arise in tonsillar crypts. It also constitutes a rare exception in the literature of carcinoma development, as carcinoma in situ (CIS) is not a recognized entity and dysplastic stages are not evident. Here we investigated the evidence of this. Hence, a systematic review and meta‐analysis was performed to study tumor origin. MEDLINE was searched and all original studies reporting tumor origin in HPV‐associated oropharyngeal cancer were included. We also used spatial transcriptomics (10× Visium) on HPV‐associated TSCC, cervical, and HPV‐independent oral cancer cases. We compared tonsillar CIS to cervical high‐grade intraepithelial lesion (HSIL). We studied epithelial cell gene expression across the sample types and epithelial cellular states. A trajectory analysis was performed in HPV‐associated tumors. In total, 14% of all TSCC were of surface origin in the systematic review. Spatial transcriptomics revealed that tonsillar CIS and HSIL clustered with a high correlation when using a pseudo‐bulk approach. Moreover, when all epithelial cells were analyzed unsupervised, ten epithelial cell clusters that correlated to histology were identified. Two of these clusters were dysplastic and occurred in all samples. A sequential progression from dysplasia to invasion was observed in both TSCC and cervical cancer in the trajectory analysis. We conclude that a subset of HPV‐associated TSCC may arise from the surface and that there is a sequential progression toward invasive disease in HPV‐associated TSCC, similar to that of cervical cancer. The findings suggest that pure tonsillar dysplastic lesions should hypothetically exist.

Chronic inflammation contributes to the development of colorectal cancer, partly through its regulation of the microenvironment and antitumor immunity. Interestingly, women have a lower incidence of colorectal cancer, and estrogen treatment has been shown to reduce the occurrence of colorectal tumors. While intestinal estrogen receptor beta (ERβ, Esr2) can protect against colitis and colitis-induced cancer in mice, its role in shaping the tumor microenvironment remains unknown. In this study, we performed RNA sequencing to analyze the transcriptome of colonic epithelia and tumors from azoxymethane/dextran sulfate sodium-treated wild-type and intestinal ERβ knockout (ERβKO^Vil) mice and vehicle-treated controls. This revealed significant differences in gene expression and enriched biological processes influenced by sex and genotype, with immune-related responses being overrepresented. Deconvolution supported differential immune cell abundance and immunostaining showed that tumors from ERβKO^Vil mice displayed significantly increased macrophage infiltration, decreased T cell infiltration, and impaired natural killer cell infiltration. Further, ERβ mRNA levels in clinical colorectal tumors correlated with immune signaling profiles and better survival. Our findings indicate that intestinal ERβ promotes an antitumor microenvironment and could potentially affect the effectiveness of immunotherapy. These insights highlight the importance of ERβ in modulating antitumor immunity and underscore its therapeutic potential in colorectal cancer.

Despite current intensive therapy, survival rates of medulloblastoma (MB) greatly vary according to molecular subgroup, so new therapies are needed. Recently, we showed that combining phosphoinositide 3-kinase (PI3K), fibroblast growth factor receptor and cyclin-dependent-kinase-4/6 inhibitors (BYL719, JNJ-42756493 and PD-0332991, respectively) or poly (ADP-ribose) polymerase (PARP) and WEE-1 inhibitors (BMN673 and MK1775 respectively) had synergistic effects on MB. Here, in continuation, we investigated the effects of single and combined administrations of PI3K and AKT inhibitors, with/without cisplatin or vincristine on adherent or suspension cultures of different MB subgroups as well as in a spheroid culture of one MB line. MB cell lines DAOY, UW228-3, D425, Med8A, and D283 were treated with single and combined administrations of BYL719, AZD5363, cisplatin or vincristine and followed for viability, cell confluence, cytotoxicity, and cell migration. DAOY was also tested as a spheroid culture. Single BYL719, AZD5363, cisplatin, or vincristine administrations gave dose-dependent responses with regard to inhibition of viability and cell confluence. Combining AZD5363 with BYL719, cisplatin or vincristine resulted in synergistic effects with regard to inhibition of viability in all cell lines, and confluence and migration in all tested cell lines. The administration of single and combined treatments to DAOY spheroids produced largely similar effects. This study provides pre-clinical evidence that AKT inhibitors combined with PI3K inhibitors, cisplatin, or vincristine exhibit additive/synergistic anti-MB activity, and lower doses could be used. The latter also applied to one MB line grown as spheroids, further supporting their future potential use.

Inflammatory intestinal conditions are a major disease burden. Numerous factors shape the distribution of immune cells in the colon, but a spatial characterization of the homeostatic and inflamed colonic immune microenvironment is lacking. Here, we use the COMET platform for multiplex immunofluorescence to profile the infiltration of nine immune cell populations in mice of both sexes (N = 16) with full spatial context, including in regions of squamous metaplasia. Unsupervised clustering, neighborhood analysis, and manual quantification along the proximal-distal axis characterized the colonic immune landscape, quantified cell-cell interactions, and revealed sex differences. The distal colon was the most affected region during colitis, which was pronounced in males, who exhibited a sex-dependent increase of B cells and reduction of M2-like macrophages. Regions of squamous metaplasia exhibited strong infiltration of numerous immune cell populations, especially in males. Females exhibited more helper T cells and neutrophils at homeostasis and increased M2-like macrophage infiltration in the mid-colon upon colitis. Sex differences were corroborated by plasma cytokine profiles. Our results provide a foundation for future studies of inflammatory intestinal conditions.