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Background: BRAF, KRAS and PIK3CA mutations are frequently found in sporadic colorectal cancer (CRC). In contrast to KRAS and PIK3CA mutations, BRAF mutations are associated with tumours harbouring CpG Island methylation phenotype (CIMP), MLH1 methylation and microsatellite instability (MSI). We aimed at determine the frequency of KRAS, BRAF and PIK3CA mutations in the process of colorectal tumourigenesis using a series of colorectal polyps and carcinomas. In the series of polyps CIMP, MLH1 methylation and MSI were also studied.Methods: Mutation analyses were performed by PCR/sequencing. Bisulfite treated DNA was used to study CIMP and MLH1 methylation. MSI was detected by pentaplex PCR and Genescan analysis of quasimonomorphic mononucleotide repeats. Chi Square test and Fisher's Exact test were used to perform association studies.Results: KRAS, PIK3CA or BRAF occur in 71% of polyps and were mutually exclusive. KRAS mutations occur in 35% of polyps. PIK3CA was found in one of the polyps. V600E BRAF mutations occur in 29% of cases, all of them classified as serrated adenoma. CIMP phenotype occurred in 25% of the polyps and all were mutated for BRAF. MLH1 methylation was not detected and all the polyps were microsatellite stable. The comparison between the frequency of oncogenic mutations in polyps and CRC (MSI and MSS) lead us to demonstrate that KRAS and PIK3CA are likely to precede both types of CRC. BRAF mutations are likely to precede MSI carcinomas since the frequency found in serrated polyps is similar to what is found in MSI CRC (P = 0.9112), but statistically different from what is found in microsatellite stable (MSS) tumours (P = 0.0191).Conclusion: Our results show that BRAF, KRAS and PIK3CA mutations occur prior to malignant transformation demonstrating that these oncogenic alterations are primary genetic events in colorectal carcinogenesis. Further, we show that BRAF mutations occur in association with CIMP phenotype in colorectal serrated polyps and verified that colorectal serrated polyps and MSI CRC show a similar frequency of BRAF mutations. These results support that BRAF mutations harbour a mild oncogenic effect in comparison to KRAS and suggest that BRAF mutant colorectal cells need to accumulate extra epigenetic alterations in order to acquire full transformation and evolve to MSI CRC. [ABSTRACT FROM AUTHOR]

Background: Low muscle mass quantity and quality (myosteatosis) can be evaluated by computed tomography (CT) by measuring skeletal muscle area and muscular attenuation, respectively, at the third lumbar vertebra. We aimed to define cut-off points of skeletal muscle area and muscular attenuation to predict mortality in non-dialysis chronic kidney disease (CKD) patients., Methods: We conducted a retrospective study including non-dialysis CKD patients over two years, who underwent an opportunistic computed tomography within a two year period, and for whom creatinine was measured within 90 days of CT. Skeletal muscle area was normalized for stature to calculate the skeletal muscle index. Area under the receiver operating characteristic (AuROC) curve and Youden's index were used, to identify the cut-point, separately according to sex., Results: One hundred sixty-seven patients (50.9% male, mean age of 68.3 ± 16.4 years) were included, most with CKD stages 3 and 4. During a median follow-up of 4.9 (4.2) years, 39 (23.4%) patients died. Muscular attenuation showed a better ability to predict mortality (AuROC curve 0.739 [95% CI 0.623-0.855] in women and 0.744 in men [95% CI 0.618-0.869]) than skeletal muscle index (AuROC curve 0.491 [95% CI 0.332-0.651] in women and 0.711 [95% CI 0.571-0.850] in men). For muscular attenuation, the best cut-off values to predict mortality were 27.56 Hounsfield units in women and 24.58 Hounsfield units in men. For skeletal muscle index, the best cut-off values were 38.47 cm 2 /m 2 in women and 47.81 cm 2 /m 2 in men. In univariable Cox-regression both low muscle mass and myosteatosis were associated with increased mortality. In multivariable Cox-regression models only myosteatosis maintained a significant association with mortality (Hazard Ratio 2.651 (95% CI 1.232-5.703, p = 0.013))., Conclusions: We found sex-specific cut-off values for muscle parameters using CT analysis in non-dialysis CKD patients that were associated with mortality. In this population, myosteatosis may be more closely associated with mortality than muscle quantity., (© 2024. The Author(s) under exclusive licence to Italian Society of Nephrology.)

Colorectal cancer (CRC) responses to KRAS-targeted inhibition have been limited due to low response rates, the mechanisms of which remain unknown. Herein, we explored the cancer-associated fibroblasts (CAFs) secretome as a mediator of resistance to KRAS silencing. CRC cell lines HCT15, HCT116, and SW480 were cultured either in recommended media or in conditioned media from a normal colon fibroblast cell line (CCD-18Co) activated with rhTGF-β1 to induce a CAF-like phenotype. The expression of membrane stem cell markers was analyzed by flow cytometry. Stem cell potential was evaluated by a sphere formation assay. RNAseq was performed in KRAS-silenced HCT116 colonospheres treated with either control media or conditioned media from CAFs. Our results demonstrated that KRAS-silencing up-regulated CD24 and down-regulated CD49f and CD104 in the three cell lines, leading to a reduction in sphere-forming efficiency. However, CAF-secreted factors restored stem cell marker expression and increased stemness. RNA sequencing showed that CAF-secreted factors up-regulated genes associated with pro-tumorigenic pathways in KRAS-silenced cells, including KRAS, TGFβ, NOTCH, WNT, MYC, cell cycle progression and exit from quiescence, epithelial-mesenchymal transition, and immune regulation. Overall, our results suggest that resistance to KRAS-targeted inhibition might derive not only from cell-intrinsic causes but also from external elements, such as fibroblast-secreted factors.

Nanostructured lipid carriers (NLCs) have the potential to increase the bioavailability and reduce the side effects of docetaxel (DTX). However, only a small fraction of nanoparticles given intravenously can reach a solid tumor. In situ-forming gels combined with nanoparticles facilitate local administration and promote drug retention at the tumor site. Injectable hydrogels based on poloxamer 407 are excellent candidates for this hybrid nanoparticle-hydrogel system because of their thermoresponsive behavior and biocompatibility. Therefore, this work aimed to develop injectable poloxamer hydrogels containing NLCs for intratumoral delivery of DTX. To ensure sterility, the obtained hydrogels were autoclaved (121 °C for 15 min) after preparation. Then, the incorporation of NLCs into the poloxamer hydrogels and the impact of steam sterilization on the nanocomposite hydrogels were evaluated concerning sol-gel transition, injectability, and physicochemical stability. All formulations were extruded through the tested syringe-needle systems with acceptable force (2.2-13.4 N) and work (49.5-317.7 N·mm) of injection. Following steam sterilization, injection became easier in most cases, and the physicochemical properties of all hydrogels remained practically unchanged according to the spectroscopical and thermal analysis. The rheological evaluation revealed that the nanocomposite hydrogels were liquid at 25 °C and underwent rapid gelation at 37 °C. However, their sterilized counterparts gelled at 1-2 °C above body temperature, suggesting that the autoclaving conditions employed had rendered these nanocomposite hydrogels unsuitable for local drug delivery.

The specific interaction between cell surface receptors and corresponding antibodies has driven opportunities for developing targeted cancer therapies using nanoparticle systems. It is challenging to design and develop such targeted nanomedicines using antibody ligands, as the final nanoconjugate's specificity hinges on the cohesive functioning of its components. The multicomponent nature of antibody-conjugated nanoparticles also complicates the characterization process. Regardless of the type of nanoparticle, it is essential to perform physicochemical characterization to establish a solid foundation of knowledge and develop suitable preclinical studies. A meaningful physicochemical evaluation of antibody-conjugated nanoparticles should include determining the quantity and orientation of the antibodies, confirming the antibodies' integrity following attachment, and assessing the immunoreactivity of the obtained nanoconjugates. In this review, the authors describe the various techniques (electrophoresis, spectroscopy, colorimetric assays, immunoassays, etc.) used to analyze the physicochemical properties of nanoparticles functionalized with antibodies and discuss the main results.

The widespread push to invest in local cancer therapies comes from the need to overcome the limitations of systemic treatment options. In contrast to intravenous administration, local treatments using intratumoral or peritumoral injections are independent of tumor vasculature and allow high concentrations of therapeutic agents to reach the tumor site with minimal systemic toxicity. Injectable biodegradable hydrogels offer a clear advantage over other delivery systems because the former requires no surgical procedures and promotes drug retention at the tumor site. More precisely, in situ gelling systems based on poloxamers have garnered considerable attention due to their thermoresponsive behavior, biocompatibility, ease of preparation, and possible incorporation of different anticancer agents. Therefore, this review focuses on the use of injectable thermoresponsive hydrogels based on poloxamers and their physicochemical and biological characterization. It also includes a summary of these hydrogel applications in local cancer therapies using chemotherapy, phototherapy, immunotherapy, and gene therapy.

Objective: We investigated the association between body composition upon diagnosis and complicated phenotypes and time until surgery in patients with Crohn's disease (CD)., Methods: We conducted a retrospective cohort study including patients with CD who had a computed tomography enterography or a magnetic resonance enterography performed ≤6 mo of diagnosis. Skeletal muscle and visceral and subcutaneous adipose tissue cross-sectional areas were determined with computed tomography or magnetic resonance images at the third lumbar vertebral level, processed with the sliceOmatic (TomoVison, Magog, QC, Canada) and ABACS plugin., Results: We included 63 patients: 33 (52%) men, median age 35 y. Disease location (L) and behavior (B) according to the Montreal classification were L1 (ileal disease) = 28 (44%), L2 (colonic disease) = 13 (21%), L3(ileocolonic disease) = 18 (28%), L1 + L4 (ileal and isolated upper disease) = 1 (2%), L3 + L4 (ileocolonic and isolated upper disease) = 3 (5%), B1 (non-stricturing) = 39 (62%), B2 (stricturing) = 11 (17%), and B3 (penetrating)= 13 (21%); 20 (32%) patients had perianal disease. Visceral obesity was present in 12 (19%) patients and was associated with higher age of CD onset (median 60 versus 34 y; P = 0.002) and complicated disease behavior (B2 and B3) (66.7% versus 31.7%; P = 0.021). After adjusting for age and perianal disease, total adipose tissue was associated with a 4% increase in the odds of complicated behavior per 10 cm 2 of total adipose tissue (odds ratio [OR] = 1.004; 95% confidence interval [CI], 1.00-1.008; P = 0.043). Median follow-up time was 3.35 y, during which 15 (24%) of patients underwent abdominal surgery. Visceral obesity was associated with 5.10-times higher risk of abdominal surgery (95% CI, 1.52-17.09; P = 0.008); after adjusting for disease behavior, visceral obesity maintained a near-significant association with a 2.90-times higher risk of surgery (95% CI, 0.83-10.08; P = 0.09)., Conclusion: Total fat was associated with complicated disease phenotype and visceral obesity, with higher risk of abdominal surgery and shorter time until surgery., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Nanotechnology takes the lead in providing new therapeutic options for cancer patients. In the last decades, lipid-based nanoparticles-solid lipid nanoparticles (SLNs), nanostructured lipid carriers (NLCs), liposomes, and lipid-polymer hybrid nanoparticles-have received particular interest in anticancer drug delivery to solid tumors. To improve selectivity for target cells and, thus, therapeutic efficacy, lipid nanoparticles have been functionalized with antibodies that bind to receptors overexpressed in angiogenic endothelial cells or cancer cells. Most papers dealing with the preclinical results of antibody-conjugated nanoparticles claim low systemic toxicity and effective tumor inhibition, which have not been successfully translated into clinical use yet. This review aims to summarize the current "state-of-the-art" in anticancer drug delivery using antibody-functionalized lipid-based nanoparticles. It includes an update on promising candidates that entered clinical trials and some explanations for low translation success.

We investigated the impactof microsatellite instability (MSI) and Epstein-Barr virus (EBV) status in gastric cancer (GC), regarding response to perioperative chemotherapy (POPChT), overall survival (OS), and progression-free survival (PFS). We included 137 cases of operated GC, 51 of which were submitted to POPChT. MSI status was determined by multiplex PCR and EBV status by EBV-encoded RNA in situ hybridization. Thirty-seven (27%) cases presented as MSI-high, and seven (5.1%) were EBV+. Concerning tumor regression after POPChT, no differences were observed between the molecular subtypes, but females were more likely to respond ( p = 0.062). No significant differences were found in OS or PFS between different subtypes. In multivariate analysis, age (HR 1.02, IC 95% 1.002-1.056, p = 0.033) and positive lymph nodes (HR 1.82, IC 95% 1.034-3.211, p = 0.038) were the only prognostic factors for OS. However, females with MSI-high tumors treated with POPChT demonstrated a significantly increased OS compared to females with MSS tumors ( p = 0.031). In conclusion, we found a high proportion of MSI-high cases. MSI and EBV status did not influence OS or PFS either in patients submitted to POPChT or surgery alone. However, superior survival of females with MSI-high tumors suggests that sex disparities and molecular classification may influence treatment options in GC.

Genetic alterations influence the malignant potential of cancer cells, and so does the tumor microenvironment. Herein, we combined the study of KRAS oncogenic effects in colorectal cancer cells with the influence of fibroblast-derived factors. Results revealed that mutant KRAS regulates cell fate through both autonomous and nonautonomous signaling mechanisms. Specifically, processes such as proliferation and cell-cell aggregation were autonomously controlled by mutant KRAS independently of the stimulation with fibroblasts conditioned media. However, cancer cell invasion revealed to be a KRAS-dependent nonautonomous effect, resulting from the cooperation between fibroblast-derived HGF and mutant KRAS regulation of C-MET expression. C-MET downregulation upon KRAS silencing rendered cells less responsive to HGF and thus less invasive. Yet, in one cell line, KRAS inhibition triggered invasion upon stimulation with fibroblasts conditioned media. Inhibition of PIK3CA oncogene did not promote invasion, thus showing a KRAS-specific effect. Moreover, the invasive capacity also depended on the HGF-C-MET axis. Overall, our study awards oncogenic KRAS an important role in modulating the response to fibroblast-secreted factors either by promoting or impairing invasion, and depicts the HGF-C-MET axis as a putative therapeutic target to impair the invasive properties of mutant KRAS cancer cells., (© 2022 UICC.)

KRAS mutations have been shown to extend their oncogenic effects beyond the cancer cell, influencing the tumor microenvironment. Herein, we studied the impact of mutant KRAS on the modulation of the pro-tumorigenic properties of cancer-associated fibroblasts (CAFs), including α-SMA expression, TGFβ1 and HGF production, extracellular matrix components and metalloproteinases expression as well as collagen contraction and migration capacities. To do so, CCD-18Co normal-like colon fibroblasts were challenged with conditioned media from control and KRAS silenced colorectal cancer (CRC) cells. Our results showed that the mutant KRAS CRC cell-secreted factors were capable of turning normal-like fibroblasts into CAF-like by modulating the α-SMA expression, TGFβ1 and HGF production and migration capacity. Oncogenic KRAS played a secondary role as its silencing did not completely impair the capacity of CRC cells to modulate most of the fibroblast properties analyzed. In summary, our work suggests that mutant KRAS does not play a major role in controlling the CRC cell-secreted factors that modulate the behavior of fibroblasts. The fact that CRC cells retain the capacity to modulate the pro-tumorigenic features of fibroblasts independently of KRAS silencing is likely to negatively impact their response to KRAS inhibitors, thus standing as a putative mechanism of resistance to KRAS inhibition with potential therapeutical relevance.

Understanding how mutant KRAS signaling is modulated by exogenous stimuli is of utmost importance to elucidate resistance mechanisms underlying pathway inhibition failure, and to uncover novel therapeutic targets for mutant KRAS patients. Hence, aiming at perceiving KRAS-autonomous versus -non autonomous mechanisms, we studied the response of two mutant KRAS colorectal cancer cell lines (HCT116 and LS174T) upon KRAS silencing and treatment with rhTGFβ1-activated fibroblasts secretome. A proteomic analysis revealed that rhTGFβ1-activated fibroblast-secreted factors triggered cell line-specific proteome alterations and that mutant KRAS governs 43% and 38% of these alterations in HCT116 and LS174T cells, respectively. These KRAS-dependent proteins were localized and displayed molecular functions that were common to both cell lines (e.g., extracellular exosome, RNA binding functions). Moreover, 67% and 78% of the KRAS-associated proteome of HCT116 and LS174T cells, respectively, was controlled in a KRAS-non-autonomous manner, being dependent on fibroblast-secreted factors. In HCT116 cells, KRAS-non-autonomously controlled proteins were mainly involved in proteoglycans in cancer, p53, and Rap1 signaling pathways; whereas in LS174T cells, they were associated with substrate adhesion-dependent cell-spreading and involved in metabolic processes. This work highlights the context-dependency of KRAS-associated signaling and reinforces the importance of integrating the tumor microenvironment in the study of KRAS-associated effects.

More than a physical structure providing support to tissues, the extracellular matrix (ECM) is a complex and dynamic network of macromolecules that modulates the behavior of both cancer cells and associated stromal cells of the tumor microenvironment (TME). Over the last few years, several efforts have been made to develop new models that accurately mimic the interconnections within the TME and specifically the biomechanical and biomolecular complexity of the tumor ECM. Particularly in colorectal cancer, the ECM is highly remodeled and disorganized and constitutes a key component that affects cancer hallmarks, such as cell differentiation, proliferation, angiogenesis, invasion and metastasis. Therefore, several scaffolds produced from natural and/or synthetic polymers and ceramics have been used in 3D biomimetic strategies for colorectal cancer research. Nevertheless, decellularized ECM from colorectal tumors is a unique model that offers the maintenance of native ECM architecture and molecular composition. This review will focus on innovative and advanced 3D-based models of decellularized ECM as high-throughput strategies in colorectal cancer research that potentially fill some of the gaps between in vitro 2D and in vivo models. Our aim is to highlight the need for strategies that accurately mimic the TME for precision medicine and for studying the pathophysiology of the disease.

The ability of some hydrogels to exhibit a phase transition or change their structure in response to stimuli has been extensively explored for drug depot formation and controlled drug release. Taking advantage of the unique features of the tumor microenvironment (TME) or externally applied triggers, several injectable stimuli-responsive hydrogels have been described as promising candidates for intratumoral drug delivery. In this review, we provide a brief overview of the TME and highlight the advantages of intratumoral administration, followed by a summary of the reported strategies to endow hydrogels with responsiveness to physical (temperature and light), chemical (pH and redox potential), or biological (enzyme) stimuli., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Ltd. All rights reserved.)