Whey-based diet containing medium chain triglycerides modulates the gut microbiota and protects the intestinal mucosa from chemotherapy while maintaining therapy efficacy.

Cytotoxicity (i.e. cell death) is the core mechanism by which chemotherapy induces its anti-cancer effects. Unfortunately, this same mechanism underpins the collateral damage it causes to healthy tissues. The gastrointestinal tract is highly susceptible to chemotherapy's cytotoxicity, resulting in ulcerative lesions (termed gastrointestinal mucositis, GI-M) that impair the functional capacity of the gut leading to diarrhea, anorexia, malnutrition and weight loss, which negatively impact physical/psychological wellbeing and treatment adherence. Preventing these side effects has proven challenging given the overlapping mechanisms that dictate chemotherapy efficacy and toxicity. Here, we report on a novel dietary intervention that, due to its localized gastrointestinal effects, is able to protect the intestinal mucosal from unwanted toxicity without impairing the anti-tumor effects of chemotherapy. The test diet (containing extensively hydrolyzed whey protein and medium chain triglycerides (MCTs)), was investigated in both tumor-naïve and tumor-bearing models to evaluate its effect on GI-M and chemo-efficacy, respectively. In both models, methotrexate was used as the representative chemotherapeutic agent and the diet was provided ad libitum for 14 days prior to treatment. GI-M was measured using the validated biomarker plasma citrulline, and chemo-efficacy defined by tumor burden (cm3/g body weight). The test diet significantly attenuated GI-M (P = 0.03), with associated reductions in diarrhea (P < 0.0001), weight loss (P < 0.05), daily activity (P < 0.02) and maintenance of body composition (P < 0.02). Moreover, the test diet showed significant impact on gut microbiota by increasing diversity and resilience, whilst also altering microbial composition and function (indicated by cecal short and brained chain fatty acids). The test diet did not impair the efficacy of methotrexate against mammary adenocarcinoma (tumor) cells. In line with the first model, the test diet minimized intestinal injury (P = 0.001) and diarrhea (P < 0.0001). These data support translational initiatives to determine the clinical feasibility, utility and efficacy of this diet to improve chemotherapy treatment outcomes.

In vitro chemotherapy-associated muscle toxicity is attenuated with nutritional support, while treatment efficacy is retained.

PURPOSE: Muscle-wasting and treatment-related toxicities negatively impact prognosis of colorectal cancer (CRC) patients. Specific nutritional composition might support skeletal muscle and enhance treatment support. In this in vitro study we assess the effect of nutrients EPA, DHA, L-leucine and vitamin D3, as single nutrients or in combination on chemotherapy-treated C2C12-myotubes, and specific CRC-tumor cells. MATERIALS AND METHODS: Using C2C12-myotubes, the effects of chemotherapy (oxaliplatin, 5-fluorouracil, oxaliplatin+5-fluorouracil and irinotecan) on protein synthesis, cell-viability, caspase-3/7-activity and LDH-activity were assessed. Addition of EPA, DHA, L-leucine and vitamin D3 and their combination (SNCi) were studied in presence of above chemotherapies. Tumor cell-viability was assessed in oxaliplatin-treated C26 and MC38 CRC cells, and in murine and patient-derived CRC-organoids. RESULTS: While chemotherapy treatment of C2C12-myotubes decreased protein synthesis, cell-viability and increased caspase-3/7 and LDH-activity, SNCi showed improved protein synthesis and cell viability and lowered LDH activity. The nutrient combination SNCi showed a better overall performance compared to the single nutrients. Treatment response of tumor models was not significantly affected by addition of nutrients. CONCLUSIONS: This in vitro study shows protective effect with specific nutrition composition of C2C12-myotubes against chemotherapy toxicity, which is superior to the single nutrients, while treatment response of tumor cells remained.

Specialized nutrition improves muscle function and physical activity without affecting chemotherapy efficacy in C26 tumour-bearing mice.

BACKGROUND: Skeletal muscle wasting and fatigue are commonly observed in cancer patients receiving chemotherapy and associated with reduced treatment outcome and quality of life. Nutritional support may mitigate these side effects, but potential interference with chemotherapy efficacy could be of concern. Here, we investigated the effects of an ω-3 polyunsaturated fatty acid (eicosapentaenoic acid and docosahexaenoic acid), leucine-enriched, high-protein (100% whey), additional vitamin D, and prebiotic fibres 'specific nutritional composition' (SNC) and chemotherapy on state-of-the-art tumour organoids and muscle cells and studied muscle function, physical activity, systemic inflammation, and chemotherapy efficacy in a mouse model of aggressive colorectal cancer (CRC). METHODS: Tumour-bearing mice received a diet with or without SNC. Chemotherapy treatment consisted of oxaliplatin and 5-fluorouracil. Tumour formation was monitored by calliper measurements. Physical activity was continuously monitored by infrared imaging. Ex vivo muscle performance was determined by myography, muscle fatty acid composition by gas chromatography, and plasma cytokine levels by Luminex xMAP technology. Patient-derived CRC organoids and C2C12 myotubes were used to determine whether SNC affects chemotherapy sensitivity in vitro. RESULTS: Specific nutritional composition increased muscle contraction capacity of chemotherapy-treated tumour-bearing mice (P < 0.05) and enriched ω-3 fatty acid composition in muscle without affecting treatment efficacy (P < 0.0001). Mice receiving SNC maintained physical activity after chemotherapy and showed decreased systemic inflammation. Therapeutic response of CRC organoids was unaffected by SNC nutrients, while cell viability and protein synthesis of muscle cells significantly improved. CONCLUSIONS: The results show that specialized nutritional support can be used to maintain muscle function and physical activity levels during chemotherapy without increasing tumour viability. Therefore, nutritional strategies have potential value in promoting cancer and chemotherapy tolerance.

The association between changes in muscle mass and quality of life in patients with metastatic colorectal cancer.

BACKGROUND: Skeletal muscle mass (SMM) loss is common in metastatic colorectal cancer (mCRC) patients and associated with poor clinical outcomes, including increased treatment-related toxicities and reduced survival. Muscle loss may contribute to reduced health-related quality of life (HRQoL), including fatigue. Our aim was to study associations between changes in SMM and concomitant changes in patient-reported HRQoL. METHODS: This was a secondary analysis of mCRC patients in the CAIRO3 randomized clinical trial who were-after initial treatment-randomized between maintenance treatment with capecitabine plus bevacizumab (CAP-B) and observation until first disease progression (PD1). Included patients had computed tomography images for SMM quantification, together with HRQoL assessments available at randomization and PD1. Changes in SMM (categorized as >2% loss, stable, and >2% gain) and HRQoL were computed between randomization and PD1. Changes in HRQoL score >10 points were considered clinically relevant. Associations between SMM and HRQoL changes were studied by multiple linear regression models. We also investigated whether associations differed by treatment arm for global health and the 13 other HRQoL subscales. RESULTS: Of 221 patients included (mean age 63.5 ± 8.4 years), 24% lost, 27% remained stable, and 49% gained SMM. At randomization, mean global health status was 73.5 ± 15.9 in the CAP-B arm and 75.1 ± 17.5 in the observation arm (P = 0.48). A stable or gain in SMM was significantly associated with a clinically relevant improvement in global health status (9.9 and 14.7 points, respectively), compared with patients who lost SMM. From the subscales that did not show significant differences between the two treatment arms, we found significant and clinically relevant associations for stable or gain in SMM with improved role functioning (12.0 and 17.9, respectively) and with less fatigue (-10.0 and -15.0, respectively) and pain (-16.3 for SMM gain). From the subscales that did show significantly different associations with SMM between the two treatment arms, we only found significant results in the observation arm. Here, associations were found for stable or gain in SMM with clinically relevant improved physical (12.4 for SMM gain), cognitive (10.7 and 9.7, respectively), and social functioning (15.5 and 15.6, respectively) as well as reduced appetite loss (-28.5 and -30.7, respectively). CONCLUSIONS: In mCRC, SMM preservation during CAP-B and observation treatment is associated with significant and clinically relevant improvements in global health status and multiple functional and symptom scales. Studies are warranted to investigate whether interventions targeting SMM lead to improved HRQoL, fewer symptoms, and better functioning.

Loss of skeletal muscle index and survival in patients with metastatic colorectal cancer: Secondary analysis of the phase 3 CAIRO3 trial.

BACKGROUND: Low skeletal muscle index (SMI) in metastatic colorectal cancer (mCRC) patients is associated with poor outcomes. The prognostic impact of SMI changes during consecutive palliative systemic treatments is unknown. METHODS: This is a retrospective analysis of the phase 3 CAIRO3 study. The CAIRO3 study randomized 557 patients between maintenance capecitabine + bevacizumab (CAP-B) or observation, after six cycles capecitabine + oxaliplatin + bevacizumab (CAPOX-B). Upon first disease progression (PD1), CAPOX-B was reintroduced until second progression (PD2). SMI was assessed by computed tomography (CT) (total 1355 scans). SMI and body mass index (BMI) changes were analyzed for three time-periods; p1: during initial CAPOX-B, p2: randomization to PD1, and p3: PD1 to PD2. The association between absolute and change in SMI and BMI (both per 1 standard deviation) during p1-p3, with PD1, PD2, and survival was studied by Cox regression models. RESULTS: This analysis included 450 of the 557 patients randomized in the CAIRO3 study. Mean SMI decreased during p1: mean -0.6 SMI units [95% CI -1.07;-0.26] and p3: -2.2 units [-2.7;-1.8], whereas during p2, SMI increased + 1.2 units [0.8-1.6]. BMI changes did not reflect changes in SMI. SMI loss during p2 and p3 was significantly associated with shorter survival (HR 1.19 [1.09-1.35]; 1.54 [1.31-1.79], respectively). Sarcopenia at PD1 was significantly associated with early PD2 (HR 1.40 [1.10-1.70]). BMI loss independent of SMI loss was only associated with shorter overall survival during p3 (HR 1.35 [1.14-1.63]). CONCLUSIONS: In mCRC patients, SMI loss during palliative systemic treatment was related with early disease progression and reduced survival. BMI did not reflect changes in SMI and could not identify patients at risk of poor outcome during early treatment lines.

DPA shows comparable chemotherapy sensitizing effects as EPA upon cellular incorporation in tumor cells.

Dietary supplementation with ω-3 polyunsaturated fatty acids (PUFAs) has been reported to enhance the sensitivity of tumor cells towards chemotherapy. Most enhancing effects are described for ω-3 PUFAs EPA and DHA; less evidence is available with the intermediate DPA. We studied the chemotherapy enhancing effects of EPA, DPA and DHA in murine colon C26 adenocarcinoma cells and showed that DPA displayed similar chemosensitizing effects as EPA. Moreover, EPA supplementation increased cellular DPA content. In a C26 tumor-bearing mouse model, we studied the incorporation of ω-3 PUFA in tumor and skeletal muscle after a diet with different ω-3 PUFA sources. Although little DPA was present in the fatty acid food sources, in those that contained considerable EPA concentrations, DPA levels were higher in tumor and muscle tissue. From these studies, we conclude that EPA and DPA show chemosensitizing effects and that intake of EPA or EPA-containing nutrition leads to increased cellular DPA content by elongation. These findings support the use of ω-3 PUFA containing nutritional supplementations in cancer patients during chemotherapy treatment.

Trajectory of body mass and skeletal muscle indices and disease progression in metastatic colorectal cancer patients.

BACKGROUND: Knowledge of the evolution of BMI and skeletal muscle index (SMI) measurements during advanced cancer and their relationships with disease progression (PD) is relevant to improve the timing of interventions that may improve cachexia-associated outcomes. OBJECTIVES: We investigated BMI and SMI trajectories and their associations with PD in metastatic colorectal cancer (mCRC) patients during consecutive palliative systemic regimens. METHODS: In a secondary analysis of the primary CAIRO3 trial, we included 533 mCRC patients with BMI measurements repeated every 3 wk and 95 randomly selected patients with SMI measurements repeated every 9 wk. We studied 2 periods: p1, during first-line maintenance capecitabine + bevacizumab or observation until the first progression of disease (PD1); and p2, during capecitabine + oxaliplatin + bevacizumab or another reintroduction treatment from PD1 until the second progression of disease (PD2). BMI and SMI trajectories were modeled separately throughout both periods, and joint longitudinal-survival modeling was used to investigate the relationships between slopes in BMI and SMI with PD at 9 and 3 wk pre-PD. A multivariate longitudinal joint model was used to investigate the association between the BMI trajectory and PD at time of PD, independent of SMI. RESULTS: During p1, the slopes in BMI and SMI were associated with early PD1 [HRs for 9-wk BMI: 1.54 (95% CI: 1.33, 1.76); 9-wk SMI: 1.38 (95% CI: 0.87, 1.89), NS; 3-wk BMI: 1.74 (95% CI: 1.48, 1.99); 3-wk SMI: 2.65 (95% CI: 1.97, 3.32)]. During p2, only the slope in SMI was related to PD2 [9-wk BMI: 1.09 (95%: CI: 0.73, 1.45), NS; 9-wk SMI: 1.64 (95% CI: 1.25, 2.04); 3-wk BMI: 1.17 (95% CI: 0.77, 1.57); 3-wk SMI: 1.11 (95% CI: 0.70, 1.53)]. In models mutually adjusting for BMI and SMI, SMI was associated with PD in p1 [p1 ( n = 95), HR BMI: 1.32 (95% CI: 0.74, 2.39), NS; p1, HR SMI: 1.50 (95% CI: 1.04, 2.14); p2 ( n = 50), BMI: 0.98 (95% CI: 0.55, 1.75), NS; p2, HR SMI: 1.11 (95% CI: 0.61, 2.05), NS]. CONCLUSIONS: In mCRC patients during palliative systemic treatment, SMI losses, irrespective of BMI losses, may be a marker for the early initiation of cachexia interventions.

Impact of different palliative systemic treatments on skeletal muscle mass in metastatic colorectal cancer patients.

BACKGROUND: Observational studies suggest that loss of skeletal muscle mass (SMM) is associated with chemotherapy-related toxicity, poor quality of life, and poor survival in metastatic colorectal cancer (mCRC) patients. Little is known about the evolution of SMM during palliative systemic therapy. We investigated changes in SMM during various consecutive palliative systemic treatment regimens using repeated abdominal computed tomography scans of mCRC patients who participated in the randomized phase 3 CAIRO3 study. METHODS: In the CAIRO3 study, mCRC patients with stable disease or better after 6 cycles of first-line treatment with capecitabine + oxaliplatin + bevacizumab (CAPOX-B) were randomized between maintenance treatment with capecitabine + bevacizumab (CAP-B) or observation. Upon first disease progression, in both groups, CAPOX-B or other treatment was reintroduced until the second disease progression, which was the primary study endpoint. We analysed 1355 computed tomography scans of 450 (81%) CAIRO3 patients (64 ± 9.0 years, CAP-B n = 223; observation n = 227) for SMM at four time points (i.e. prior to the start of pre-randomization initial treatment, at randomization, and at first and at second disease progression) using the Slice-o-matic software and single slice evaluation at the lumbar 3 level. By using accepted and widely used formulas, whole body SMM was calculated. A linear mixed effects model, adjusted for relevant confounders, was used to assess SMM changes for the total group and within and between study arms. RESULTS: During 6 cycles of initial treatment with CAPOX-B prior to randomization, SMM decreased significantly in all patients [CAP-B arm: -0.53 kg (95% CI -1.12; -0.07) and observation arm: -0.85 kg (-1.45; -0.25)]. After randomization, SMM recovered during CAP-B treatment by 1.32 kg (0.73; 1.90) and observation by 1.20 kg (0.63; 1.78) (median time from randomization to first disease progression 8.6 and 4.1 months for CAP-B arm and observation arm, respectively). After first progression and during reintroduction treatment with CAPOX-B or other treatment, SMM again decreased significantly and comparable in both arms, CAP-B: -2.71 kg (-3.37; -2.03), and observation: -2.01 kg (-2.64; -1.41) (median time from first progression until second progression CAP-B arm: 4.7 months and observation arm: 6.6 months). CONCLUSIONS: This longitudinal study provides a unique insight in SMM changes in mCRC patients during palliative systemic treatment regimens, including observation. Our data show that muscle loss is reversible and may be influenced by the intensity of systemic regimens. Although studies have shown prognostic capacity for SMM, the effects of subsequent changes in SMM are unknown and may be clues for new future therapeutic interventions.

Hypoxia-Targeting Fluorescent Nanobodies for Optical Molecular Imaging of Pre-Invasive Breast Cancer.

PURPOSE: The aim of this work was to develop a CAIX-specific nanobody conjugated to IRDye800CW for molecular imaging of pre-invasive breast cancer. PROCEDURES: CAIX-specific nanobodies were selected using a modified phage display technology, conjugated site-specifically to IRDye800CW and evaluated in a xenograft breast cancer mouse model using ductal carcinoma in situ cells (DCIS). RESULTS: Specific anti-CAIX nanobodies were obtained. Administration of a CAIX-specific nanobody into mice with DCIS xenografts overexpressing CAIX showed after 2 h a mean tumor-to-normal tissue ratio (TNR) of 4.3 ± 0.6, compared to a TNR of 1.4 ± 0.2 in mice injected with the negative control nanobody R2-IR. In DCIS mice, a TNR of 1.8 ± 0.1 was obtained. Biodistribution studies demonstrated an uptake of 14.0 ± 1.1 %I.D./g in DCIS + CAIX tumors, 4.6 ± 0.8 %I.D./g in DCIS tumors, while 2.0 ± 0.2 %I.D./g was obtained with R2-IR. CONCLUSIONS: These results demonstrate the successful generation of a CAIX-specific nanobody-IRDye800CW conjugate that can be used for rapid imaging of (pre-)invasive breast cancer.

Camelid heavy chain only antibody fragment domain against ß-site of amyloid precursor protein cleaving enzyme 1 inhibits ß-secretase activity in vitro and in vivo.

UNLABELLED: Accumulation and aggregation of the amyloid-ß (Aß) peptide is associated with Alzheimer's disease (AD). Aß is generated from the amyloid precursor protein by the successive action of two membrane-associated processing enzymes: ß-secretase or ß-site of amyloid precursor protein cleaving enzyme 1 (BACE1) and γ-secretase. Inhibition of one or both of these enzymes prevents Aß generation and the accompanying Aß accumulation. Antigen binding fragments from camelid heavy chain only antibodies (VHHs) were found to exert excellent enzyme inhibition activity. In the present study, we generated VHHs against BACE1 by active immunization of Lama glama with the recombinant BACE1 protein. Two classes of VHHs were selected from a VHH-phage display library by competitive elution with a peptide encoding the Swedish mutation variant of the BACE1 processing site. One VHH was found to inhibit the enzyme activity of BACE1 in vitro and in cell culture, whereas two other VHHs were found to stimulate BACE1 activity under the same conditions in vitro. Furthermore, an in vivo study with a transgenic AD mouse model, using intracisternal injection of the inhibitory VHH, led to acute reduction of the Aß load in the blood and brain. This inhibitory VHH may be considered as a candidate molecule for a therapy directed towards reduction of Aß load and prevention of AD progression. Both the inhibitory and stimulatory VHH may be useful for improving our understanding of the structure-function relationship of BACE1, as well as its role in AD progression. DATABASE: The GenBank sequence accession numbers are KR363186 for VHH B1a; KR363187 for VHH B3a; and KR363188 for VHH B5a.

Nanobody-based cancer therapy of solid tumors.

The development of tumor-targeted therapies using monoclonal antibodies has been successful during the last 30 years. Nevertheless, the efficacy of antibody-based therapy is still limited and further improvements are eagerly awaited. One of the promising novel developments that may overcome the drawbacks of monoclonal antibody-based therapies is the employment of nanobodies. Current nanobody-based therapeutics can be divided into three different platforms with nanobodies functioning as: receptor antagonists; targeting moieties of effector domains; or targeting molecules on the surface of nanoparticles. In this article, we describe factors that affect their performance at three different stages: their systemic circulation upon intravenous injection; their extravasation and tumor penetration; and, finally, their interaction with target molecules.

Etiological role of viruses in outbreaks of acute gastroenteritis in The Netherlands from 1994 through 2005.

Acute gastroenteritis is one of the most common diseases worldwide. In developed countries, viruses, particularly noroviruses, are recognized as the leading cause. In The Netherlands, the surveillance of gastroenteritis outbreaks with suspected viral etiologies (as determined by Kaplan criteria) was established by the National Institute for Public Health and the Environment in 1994. This paper presents an overview of viral gastroenteritis outbreaks reported from 1994 through 2005. A minimum epidemiological data set consisting of the associated setting(s), the probable transmission mode, the date of the first illness and the date of sampling, the number of persons affected, and the number of hospitalizations was requested for each reported outbreak. Stool samples were tested for the presence of norovirus, sapovirus, rotavirus, astrovirus, adenovirus, and Aichi virus by electron microscopy, enzyme-linked immunosorbent assay, and/or reverse transcription-PCR. A total of 6,707 stool samples from 941 gastroenteritis outbreaks were investigated. Noroviruses were detected as the causative agent in 735 (78.1%) of the outbreaks, and rotaviruses, adenoviruses, and astroviruses were found to be responsible for 46 (4.9%), 9 (1.0%), and 5 (0.5%) outbreaks, respectively. Among the gastroenteritis outbreaks in which a mode of transmission was identified, most outbreaks (38.1%) were associated with person-to-person transmission, and the majority (54.9%) of the outbreaks investigated were reported by residential institutions. Since 2002, the total number of outbreaks reported and the number of unexplained outbreaks have increased. Furthermore, the number of rotavirus-associated outbreaks has increased, especially in nursing homes. Despite thorough testing, 115 (12.2%) outbreaks suspected of having viral etiologies remain unexplained. Increases in numbers of reported outbreaks may indicate undefined changes in the criteria for reporting or the emergence of new pathogens.