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Obesity and metabolic dysfunction are implicated in colorectal cancer development. Appetite-regulating gut hormones might have a role in colorectal cancer risk. We investigated whether circulating levels of the gut hormones ghrelin (analyzed as acyl ghrelin) and Peptide YY (PYY) were associated with subsequent colorectal cancer risk, including clinical and molecular tumor subtypes. We also provide descriptive data on these hormones in relation to background participant characteristics and metabolic biomarkers. This population-based study included 1,010 matched case-control pairs with a median of 12.3 years of follow-up. Acyl ghrelin and PYY were measured by multiplex immunoassay. Data on KRAS and BRAF mutations and microsatellite instability (MSI) status were available for 704 and 708 cases, respectively. Conditional logistic regression models estimated association to colorectal cancer risk. Partial correlation and linear regression were used to investigate relationships between background and metabolic variables and variation in plasma gut hormone concentrations. Acyl ghrelin was not clearly associated with colorectal cancer risk (multivariable OR per 1 SD increase: 1.11; 95% CI, 1.00-1.23). Positive associations were observed for specific subtypes, in particular BRAF-mutated colorectal cancer and right-sided colon cancer, although with nonsignificant heterogeneity. PYY was not related to colorectal cancer risk (multivariable OR per 1 SD: 1.04; 95% CI, 0.95-1.14) or any tumor subtype. In the control participants, ghrelin was inversely correlated with BMI, and PYY was positively correlated with C-peptide and insulin levels. These findings provide limited support for a possible role for ghrelin in colorectal cancer development, primarily in specific anatomical and molecular tumor subtypes. PREVENTION RELEVANCE: The findings of this study do not support a major role for the metabolic gut hormones ghrelin and PYY in colorectal cancer development but suggest the possibi

Increasing evidence suggests that the gut microbiota may impact colorectal cancer (CRC) development and progression. In this study, the tumour colonisation of two CRC-associated bacteria, Parvimonas micra and Fusobacterium nucleatum, was studied in relation to patient survival in a cohort of 257 CRC patients. Colonisation of P. micra and F. nucleatum was analysed in fresh frozen tumour tissue (n = 112) and in faeces (n = 250) by qPCR. When analysing tumour tissues, both P. micra and F. nucleatum were found to be associated with decreased five-year cancer-specific survival, an association that remained significant in multivariable analysis for P. micra. Furthermore, we found significant associations of high levels of P. micra and F. nucleatum with tumour molecular characteristics, i.e., tumours mutated in BRAFV600E, and tumours of the MSI subtype. The analysis of faecal samples showed weaker associations with prognosis and tumour molecular characteristics. In conclusion, our findings support a novel association of tumour colonisation of P. micra with decreased patient survival. A better understanding of the role of the gut microbiota in CRC might contribute to the advancement of prognostic tools and new targets for therapy.

Colorectal cancer (CRC) is a heterogeneous disease with different genetic and molecular backgrounds, leading to a diverse patient prognosis and treatment response. Four consensus molecular subtypes (CMS 1–4) have recently been proposed based on transcriptome profiling. A clinically practical immunohistochemistry (IHC) based CMS classifier consisting of the four markers FRMD6, ZEB1, HTR2B, and CDX2 was then demonstrated. However, the IHC-CMS classifier did not distinguish between CMS2 and CMS3 tumours. In this study, we have applied the proposed transcriptome based and IHC-based CMS classifiers in a CRC cohort of 65 patients and found a concordance of 77.5 %. Further, we modified the IHC-CMS classifier by analysing the differentially expressed genes between CMS2 and CMS3 tumours using RNA-sequencing data from the TCGA dataset. The result showed that WNT signalling was among the most upregulated pathways in CMS2 tumours, and the expression level of CTNNB1 (encoding β-catenin), a WNT pathway hallmark, was significantly upregulated (P = 1.15 × 10−6). We therefore introduced nuclear β-catenin staining to the IHC-CMS classifier. Using the modified classifier in our cohort, we found a 71.4 % concordance between the IHC and RNA-sequencing based CMS classifiers. Moreover, β-catenin staining could classify 16 out of the 19 CMS2/3 tumours into CMS2 or CMS3, thereby showing an 84.2 % concordance with the RNA-sequencing-based classifier. In conclusion, we evaluated CMS classifiers based on transcriptome and IHC analysis. We present a modified IHC panel that categorizes CRC tumours into the four CMS groups. To our knowledge, this is the first study using IHC to identify all four CMS groups.

The local anti-tumour immune response has important prognostic value in colorectal cancer (CRC). In the era of immunotherapy, a better understanding of the immune response in molecular subgroups of CRC may lead to significant advances in personalised medicine. On this note, microsatellite instable (MSI) tumours have been characterised by increased immune infiltration, suggesting MSI as a marker for immune inhibitor checkpoint therapy. Here, we used flow cytometry to perform a comprehensive analysis of immune activity profiles in tumour tissues, adjacent non-malignant tissues and blood, from a cohort of 69 CRC patients. We found several signs of immune suppression in tumours compared to adjacent non-malignant tissues, including T cells more often expressing the immune checkpoint molecules programmed cell death protein (PD-1) and cytotoxic T lymphocyte-associated protein 4 (CTLA-4). We further analysed immune cell infiltration in molecular subgroups of CRC. MSI tumours were indeed found to be associated with increased immune infiltration, including increased fractions of PD-1+ T cells. No correlation was, however, found between MSI and the fraction of CTLA-4+ T cells. Interestingly, within the group of patients with microsatellite stable (MSS) tumours, some also presented with increased immune infiltration, including comparably high portions of PD-1+ T cells, but also CTLA-4+ T cells. Furthermore, no correlation was found between PD-1+ and CTLA-4+ T cells, suggesting that different tumours may, to some extent, be regulated by different immune checkpoints. We further evaluated the distribution of immune activity profiles in the consensus molecular subtypes of CRC. In conclusion, our findings suggest that different immune checkpoint inhibitors may be beneficial for selected CRC patients irrespective of MSI status. Improved predictive tools are required to identify these patients.

Background: Inflammation has been implicated in colorectal cancer etiology, but the relationship between C-reactive protein (CRP) and colorectal cancer risk is unclear. We aimed to investigate the association between prediagnostic plasma CRP concentrations and the risk of clinical and molecular colorectal cancer subtypes. Methods: We used prospectively collected samples from 1,010 matched colorectal cancer case-control pairs from two population-based cohorts in Northern Sweden, including 259 with repeated samples. Conditional logistic regression and linear mixed models were used to estimate relative risks of colorectal cancer, including subtypes based on BRAF and KRAS mutations, microsatellite instability status, tumor location, stage, lag time, and (using unconditional logistic regression) body mass index. Results: CRP was not associated with colorectal cancer risk, regardless of clinical or molecular colorectal cancer subtype. For participants with advanced tumors and blood samples <5 years before diagnosis, CRP was associated with higher risk [OR per 1 unit increase in natural logarithm (In) transformed CRP, 1.32; 95% confidence interval (CI), 1.01-1.73]. CRP levels increased over time, but average time trajectories were similar for cases and controls (P-interaction = 0.19). Conclusions: Our results do not support intertumoral heterogeneity as an explanation for previous inconsistent findings regarding the role of CRP in colorectal cancer etiology. The possible association in the subgroup with advanced tumors and shorter follow-up likely reflects undiagnosed cancer at baseline. Impact: Future efforts to establish the putative role of chronic, low-grade inflammation in colorectal cancer development will need to address the complex relationship between systemic inflammatory factors and tumor microenvironment, and might consider larger biomarker panels than CRP alone.

The use of faecal microbial markers as non-invasive biomarkers for colorectal cancer (CRC) has been suggested, but not fully elucidated. Here, we have evaluated the importance of Parvimonas micra as a potential non-invasive faecal biomarker in CRC and its relation to other microbial biomarkers. The levels of P. micra, F. nucleatum and clbA+bacteria were quantified using qPCR in faecal samples from a population-based cohort of patients undergoing colonoscopy due to symptoms from the large bowel. The study included 38 CRC patients, 128 patients with dysplasia and 63 controls. The results were validated in a second consecutive CRC cohort including faecal samples from 238 CRC patients and 94 controls. We found significantly higher levels of P. micra in faecal samples from CRC patients compared to controls. A test for P. micra could detect CRC with a specificity of 87.3% and a sensitivity of 60.5%. In addition, we found that combining P. micra with other microbial markers, could further enhance test sensitivity. Our findings support the potential use of P. micra as a non-invasive biomarker for CRC. Together with other microbial faecal markers, P. micra may identify patients with "high risk" microbial patterns, indicating increased risk and incidence of cancer.

Colorectal cancer (CRC) is a heterogeneous disease, with varying clinical presentations and patient prognosis. Different molecular subgroups of CRC should be treated differently and therefore, must be better characterized. Organoid culture has recently been suggested as a good model to reflect the heterogeneous nature of CRC. However, organoid cultures cannot be established from all CRC tumors. The study examines which CRC tumors are more likely to generate organoids and thus benefit from ex vivo organoid drug testing. Long-term organoid cultures from 22 out of 40 CRC tumor specimens were established. It was found that organoid cultures were more difficult to establish from tumors characterized as microsatellite instable (MSI), BRAF-mutated, poorly differentiated and/or of a mucinous type. This suggests that patients with such tumors are less likely to benefit from ex vivo organoid drug testing, but it may also suggest biological difference in tumor growth. RNA sequencing analysis of tumor sections revealed that the in vivo maintenance of these non-organoid-forming tumors depends on factors related to inflammation and pathogen exposure. Furthermore, using TCGA data we could show a trend towards a worse prognosis for patients with organoid-forming tumors, suggesting also clinical differences. Results suggest that organoids are more difficult to establish from tumors characterized as MSI, BRAF-mutated, poorly differentiated and/or of a mucinous type. We further suggest that the maintenance of cell growth of these tumors in vivo may be promoted by immune-related factors and other stromal components within the tumor microenvironment.

Factors related to energy metabolism and the metabolic syndrome, such as higher body mass index (BMI), blood glucose, or blood lipids, and blood pressure, are associated with an increased risk of colorectal cancer (CRC). However, CRC is a heterogeneous disease, developing through distinct pathways with differences in molecular characteristics and prognosis, and possibly also in risk factors. For subtypes defined by KRAS and BRAF mutation status, BMI is the only metabolic factor previously studied, with inconsistent findings. We investigated whether associations between BMI, blood glucose, blood lipids, and blood pressure and CRC risk differed by tumor KRAS and BRAF mutation status in 117,687 participants from two population-based cohorts within the Northern Sweden Health and Disease Study (NSHDS). Hazard ratios (HRs) for overall CRC and CRC subtypes by metabolic factors were estimated with Cox proportional hazards regression, using multiple imputation to handle missing exposure and tumor data. During a median follow-up of 15.6 years, we acquired 1,250 prospective CRC cases, of which 766 cases had complete baseline and molecular tumor data. Consistent with previous evidence, higher BMI, total cholesterol, triglyceride levels, and blood pressure were associated with an increased risk of overall CRC (HRs per 1 standard deviation increase: 1.07 to 1.12). These associations were similar regardless of CRC subtype by KRAS and BRAF mutation status (all p(heterogeneity) > 0.05). The same was true for subtypes based on microsatellite instability status. Poor metabolic health may therefore be a universal mechanism for colorectal cancer, acting across multiple developmental pathways.

Background/Aim: KRAS and BRAF are two genes commonly mutated in colorectal cancer (CRC). Even though BRAF is a downstream target of KRAS in the MAPK signalling pathway, KRAS- and BRAF-mutated CRCs are found to display several different clinical and histopathological features. We investigated whether a differential expression of microRNAs (miRNAs) could explain the clinicopathological differences seen between KRAS-and BRAF-mutated CRCs. Materials and Methods: Using a PCR array, we analyzed the expression of 84 different miRNAs in CRC cell lines wild-type in KRAS and BRAF, or mutated in KRAS or BRAF. Results: Ten miRNAs were selected for further analyses in tumor tissue specimens (let-7a, let-7i, miR-10a, miR-10b, miR-31, miR-100, miR-181a, miR-181b, miR-372, and miR-373). BRAF-mutated tumors were found to express significantly higher levels of miR-31 as well as significantly lower levels of miR-373, compared to wild-type tumors. Conclusion: Our results suggest that KRAS and BRAF-mutated CRCs may have different miRNA signatures compared to CRC tumors wild-type in KRAS and BRAF. However, no difference in expression levels between KRAS-and BRAF-mutated tumors was evident for the miRNAs analyzed in this study., Originally included in thesis in manuscript form

Francisella bacteria cause severe disease in both vertebrates and invertebrates and include one of the most infectious human pathogens. Mammalian cell lines have mainly been used to study the mechanisms by which Francisella manipulates its host to replicate within a large variety of hosts and cell types, including macrophages. Here, we describe the establishment of a genetically and biochemically tractable infection model: the amoeba Dictyostelium discoideum combined with the fish pathogen Francisella noatunensis subsp. noatunensis. Phagocytosed F. noatunensis subsp. noatunensis interacts with the endosomal pathway and escapes further phagosomal maturation by translocating into the host cell cytosol. F. noatunensis subsp. noatunensis lacking IglC, a known virulence determinant required for Francisella intracellular replication, follows the normal phagosomal maturation and does not grow in Dictyostelium. The attenuation of the F. noatunensis subsp. noatunensis Delta iglC mutant was confirmed in a zebrafish embryo model, where growth of F. noatunensis subsp. noatunensis Delta iglC was restricted. In Dictyostelium, F. noatunensis subsp. noatunensis interacts with the autophagic machinery. The intracellular bacteria colocalize with autophagic markers, and when autophagy is impaired (Dictyostelium Delta atg1), F. noatunensis subsp. noatunensis accumulates within Dictyostelium cells. Altogether, the Dictyostelium-F. noatunensis subsp. noatunensis infection model recapitulates the course of infection described in other host systems. The genetic and biochemical tractability of the system allows new approaches to elucidate the dynamic interactions between pathogenic Francisella and its host organism.

The bacterium Francisella noatunensis ssp. noatunensis (in text: F. noatunensis) is the ethiological agent of the disease francisellosis in Atlantic cod. Francisellosis has been one of the major limiting factors in the development of Norwegian aquaculture industry based on Atlantic cod. Lacking an effective treatment or vaccine there is urgent need for studies related to the pathogenesis of the disease. The closely related human pathogen F. tularensis is more extensively studied and due to relatively high sequence similarity with F. noatunensis, indirect evidence on important virulence factors can be obtained by reverse genetics. The Francisella Pathogenicity Island (FPI) has been identified in all sequenced genomes of Francisella sp. and contains genes associated with the ability of the bacterium to survive and replicate within macrophages. To elucidate the pathogenesis of F. noatunensis, infection assays have been performed on primary cells extracted from the head kidney of Atlantic cod. Disruptive mutations of the potential virulence factors IglC, IglD (important for intracellular growth in F. tularensis subsp.) and ClpB (a heat shock protein identified in F. tularensis), have been constructed in F. noatunensis and the infection pattern is in the process of characterization. Model systems that are utilized in the characterization are the amoebae and professional phagocyte Dictyostelium discoideum, zebrafish and macrophages extracted from head kidney of Atlantic cod.

Tularemia or vaccination with the live vaccine strain (LVS) of Francisella tularensis confers long-lived cell-mediated immunity. We hypothesized that this immunity depends on polyfunctional memory T cells, i.e., CD4(+) and/or CD8(+) T cells with the capability to simultaneously express several functional markers. Multiparametric flow cytometry, measurement of secreted cytokines, and analysis of lymphocyte proliferation were used to characterize in vitro recall responses of peripheral blood mononuclear cells (PBMC) to killed F. tularensis antigens from the LVS or Schu S4 strains. PBMC responses were compared between individuals who had contracted tularemia, had been vaccinated, or had not been exposed to F. tularensis (naive). Significant differences were detected between either of the immune donor groups and naive individuals for secreted levels of IL-5, IL-6, IL-10, IL-12, IL-13, IFN-gamma, MCP-1, and MIP-1 beta. Expression of IFN-gamma, MIP-1 beta, and CD107a by CD4(+)CD45RO(+) or CD8(+) CD45RO(+) T cells correlated to antigen concentrations. In particular, IFN-gamma and MIP-1 beta strongly discriminated between immune and naive individuals. Only one cytokine, IL-6, discriminated between the two groups of immune individuals. Notably, IL-2- or TNF-alpha-secretion was low. Our results identify functional signatures of T cells that may serve as correlates of immunity and protection against F. tularensis.

Francisella tularensis subspecies tularensis is a highly virulent facultative intracellular pathogen of humans and a potential biological weapon. A live vaccine strain, F. tularensis LVS, was developed more than 50 years ago by pragmatic attenuation of a strain of the less virulent holarctica subspecies. LVS was demonstrated to be highly effective in human volunteers who were exposed to intradermal challenge with fully virulent subsp. tularensis, but was less effective against aerosol exposure. LVS faces regulatory hurdles that to date have prevented its licensure for general use. Therefore, a better defined and more effective vaccine is being sought. To this end we have created gene deletion mutants in the virulent subsp. tularensis strain and tested them for their ability to elicit a protective immune response against systemic or aerosol challenge with the highly virulent wild-type subsp. tularensis strain, SCHU S4. Both oral and intradermal (ID) primary vaccination routes were assessed in BALB/c and C3H/HeN mice as was oral boosting. One SCHU S4 mutant missing the heat shock gene, clpB, was significantly more attenuated than LVS whereas a double deletion mutant missing genes FTT0918 and capB was as attenuated as LVS. In general mice immunized with SCHU S4DeltaclpB were significantly better protected against aerosol challenge than mice immunized with LVS. A single ID immunization of BALB/c mice with SCHU S4DeltaclpB was at least as effective as any other regimen examined. Mice immunized with SCHU S4Delta0918DeltacapB were generally protected to a similar degree as mice immunized with LVS. A preliminary examination of immune responses to vaccination with LVS, SCHU S4DeltaclpB, or SCHU S4Delta0918DeltacapB provided no obvious correlate to their relative efficacies.

A disadvantage of several old vaccines is that the genetic events resulting in the attenuation are often largely unknown and reversion to virulence cannot be excluded. In the 1950s, a live vaccine strain, LVS, was developed from a type B strain of Francisella tularensis, the causative agent of tularemia. LVS, which is highly attenuated for humans but still virulent for mice by some infection routes, has been extensively studied and found to protect staff from laboratory-acquired tularemia. The efforts to improve biopreparedness have identified a demand for a vaccine against tularemia. Recently the rapid progress in genomics of different Francisella strains has led to identification of several regions of differences (RDs). Two genes carried within RDs, pilA, encoding a putative type IV pilin, and FTT0918, encoding an outer membrane protein, have been linked to virulence. Interestingly, LVS has lost these two genes via direct repeat-mediated deletions. Here we show that reintroduction of the two deleted regions restores virulence of LVS in a mouse infection model to a level indistinguishable from that of virulent type B strains. The identification of the two attenuating deletion events could facilitate the licensing of LVS for use in humans.

BACKGROUND: Francisella tularensis is a highly virulent human pathogen. The most virulent strains belong to subspecies tularensis and these strains cause a sometimes fatal disease. Despite an intense recent research effort, there is very limited information available that explains the unique features of subspecies tularensis strains that distinguish them from other F. tularensis strains and that explain their high virulence. Here we report the use of targeted mutagenesis to investigate the roles of various genes or pathways for the virulence of strain SCHU S4, the type strain of subspecies tularensis. METHODOLOGY/PRINCIPAL FINDINGS: The virulence of SCHU S4 mutants was assessed by following the outcome of infection after intradermal administration of graded doses of bacteria. By this route, the LD(50) of the SCHU S4 strain is one CFU. The virulence of 20 in-frame deletion mutants and 37 transposon mutants was assessed. A majority of the mutants did not show increased prolonged time to death, among them notably Delta pyrB and Delta recA. Of the remaining, mutations in six unique targets, tolC, rep, FTT0609, FTT1149c, ahpC, and hfq resulted in significantly prolonged time to death and mutations in nine targets, rplA, wbtI, iglB, iglD, purL, purF, ggt, kdtA, and glpX, led to marked attenuation with an LD(50) of > 10(3) CFU. In fact, the latter seven mutants showed very marked attenuation with an LD(50) of > or = 10(7) CFU. CONCLUSIONS/SIGNIFICANCE: The results demonstrate that the characterization of targeted mutants yielded important information about essential virulence determinants that will help to identify the so far little understood extreme virulence of F. tularensis subspecies tularensis.

Francisella tularensis is a facultative intracellular bacterial pathogen capable of proliferating within host macrophages. The mechanisms that explain the differences in virulence between various strains of the species are not well characterized. In the present study, we show that both attenuated (strain LVS) and virulent (strains FSC200 and SCHU S4) strains of the pathogen replicate at similar rates in resting murine peritoneal exudate cells (PEC). However, when PEC were activated by exposure to gamma interferon (IFN-gamma), they killed LVS more rapidly than virulent strains of the pathogen. Addition of N(G)-monomethyl-l-arginine, an inhibitor of inducible nitric oxide synthase, to IFN-gamma-treated PEC, completely inhibited killing of the virulent strains, whereas it only partially blocked the killing of LVS. Similarly, in a cell-free system, SCHU S4 and FSC200 were more resistant to killing by H(2)O(2) and ONOO(-) than F. tularensis LVS. Catalase encoded by katG is a bacterial factor that can detoxify bactericidal compounds such as H(2)O(2) and ONOO(-). To investigate its contribution to the virulence of F. tularensis, katG deletion-containing mutants of SCHU S4 and LVS were generated. Both mutants demonstrated enhanced susceptibility to H(2)O(2) in vitro but replicated as effectively as the parental strains in unstimulated PEC. In mice, LVS-DeltakatG was significantly attenuated compared to LVS whereas SCHU S4-DeltakatG, despite slower replication, killed mice as quickly as SCHU S4. This implies that clinical strains of the pathogen have katG-independent mechanisms to combat the antimicrobial effects exerted by H(2)O(2) and ONOO(-), the loss of which could have contributed to the attenuation of LVS.

Background: Body fatness measured as high body mass index (BMI) increase the risk for colorectal cancer (CRC). The mechanisms behind the relationship are not fully understood, but might include insulin resistance and changes in adipokine concentrations produced by adipose tissue. Yet, associations between circulating biomarkers related to these mechanisms and CRC risk have been somewhat inconsistent, possibly due to CRC heterogeneity. To better understand the role of insulin resistance and adipokines in CRC development, we therefore investigated circulating biomarkers related to these mechanisms in relation to molecular subtypes of CRC. Methods: This was a prospective case-control study of 1010 cases and 1:1 matched controls nested within the population-based Northern Sweden Health and Disease Study (NSHDS). Concentrations of insulin, C-peptide, adiponectin, and leptin were quantified in prediagnostic plasma using immunoassays and related to CRC and CRC subtypes defined by mutations in BRAF and KRAS, and microsatellite instability (MSI) status analyzed in tumor tissue. Odds ratios (ORs) and 95% confidence intervals (CIs) for CRC by metabolic biomarker levels were calculated with conditional logistic regression. Results: Higher C-peptide and lower adiponectin were associated with an increased CRC risk (ORs per 1 standard deviation increase (95% CI): 1.11 (1.01, 1.23) and 0.91 (0.83, 1.00), respectively). The associations were attenuated when adjusting for BMI (ORs (95% CI): 1.07 (0.96, 1.19) and 0.93 (0.84, 1.03), respectively), with the potential exception of the association of C-peptide in women. Circulating insulin and leptin were not associated with CRC risk. We found no clear differences in the association between any biomarkers and CRC risk by molecular subtypes defined by KRAS and BRAF mutation status (Pheterogeneity>0.6), or MSI status (Pheterogeneity>0.3). Conclusion: Circulating biomarkers of insulin resistance and adipokines were not associated with