Therapeutic vaccination with lentiviral vector in HBV-persistent mice and two inactive HBsAg carriers.

BACKGROUND & AIMS: Immunotherapy for chronic hepatitis B virus (HBV) infection has not yet demonstrated sufficient efficacy. We developed a non-integrative lentiviral-vectored therapeutic vaccine for chronic hepatitis B and tested its antiviral effects in HBV-persistent mice and two inactive HBsAg carriers. METHODS: Lentiviral vectors (LVs) encoding the core, preS1, or large HBsAg (LHBs) proteins of HBV were evaluated for immunogenicity in HBV-naïve mice and therapeutic efficacy in a murine model of chronic HBV infection. In addition, two inactive HBsAg carriers each received two doses of 5×107 transduction units (TU) or 1×108 TU of lentiviral-vectored LHBs (LV-LHBs), respectively. The endpoints were safety, LHBs-specific T-cell responses, and serum HBsAg levels during a 24-week follow-up. RESULTS: In the mouse models, LV-LHBs was the most promising in eliciting robust antigen-specific T cells and in reducing the levels of serum HBsAg and viral load. By the end of the 34-week observation period, six out of ten (60%) HBV-persistent mice vaccinated with LV-LHBs achieved serum HBsAg loss and significant depletion of HBV-positive hepatocytes in the liver. In the two inactive HBsAg carriers, vaccination with LV-LHBs induced a considerable increase in the number of peripheral LHBs-specific T cells in one patient, and a weak but detectable response in the other, accompanied by a sustained reduction of HBsAg (-0.31 log10 IU/ml and -0.46 log10 IU/ml, respectively) from baseline to nadir. CONCLUSIONS: A lentiviral-vectored therapeutic vaccine for chronic HBV infection demonstrated the potential to improve HBV-specific T-cell responses and deplete HBV-positive hepatocytes, leading to a sustained loss or reduction of serum HBsAg. IMPACT AND IMPLICATIONS: Chronic HBV infection is characterized by an extremely low number and profound hypo-responsiveness of HBV-specific T cells. Therapeutic vaccines are designed to improve HBV-specific T-cell responses. We show that immunization with a lentiviral-vectored therapeutic HBV vaccine was able to expand HBV-specific T cells in vivo, leading to reductions of HBV-positive hepatocytes and serum HBsAg.

A mechanism by which gut microbiota elevates permeability and inflammation in obese/diabetic mice and human gut.

OBJECTIVE: Ample evidence exists for the role of abnormal gut microbiota composition and increased gut permeability ('leaky gut') in chronic inflammation that commonly co-occurs in the gut in both obesity and diabetes, yet the detailed mechanisms involved in this process have remained elusive. DESIGN: In this study, we substantiate the causal role of the gut microbiota by use of faecal conditioned media along with faecal microbiota transplantation. Using untargeted and comprehensive approaches, we discovered the mechanism by which the obese microbiota instigates gut permeability, inflammation and abnormalities in glucose metabolism. RESULTS: We demonstrated that the reduced capacity of the microbiota from both obese mice and humans to metabolise ethanolamine results in ethanolamine accumulation in the gut, accounting for induction of intestinal permeability. Elevated ethanolamine increased the expression of microRNA-miR-101a-3p by enhancing ARID3a binding on the miR promoter. Increased miR-101a-3p decreased the stability of zona occludens-1 (Zo1) mRNA, which in turn, weakened intestinal barriers and induced gut permeability, inflammation and abnormalities in glucose metabolism. Importantly, restoring ethanolamine-metabolising activity in gut microbiota using a novel probiotic therapy reduced elevated gut permeability, inflammation and abnormalities in glucose metabolism by correcting the ARID3a/miR-101a/Zo1 axis. CONCLUSION: Overall, we discovered that the reduced capacity of obese microbiota to metabolise ethanolamine instigates gut permeability, inflammation and glucose metabolic dysfunctions, and restoring ethanolamine-metabolising capacity by a novel probiotic therapy reverses these abnormalities. TRIAL REGISTRATION NUMBER: NCT02869659 and NCT03269032.

Enteric Delivery of Regenerating Family Member 3 alpha Alters the Intestinal Microbiota and Controls Inflammation in Mice With Colitis.

BACKGROUND & AIMS: Paneth cell dysfunction causes deficiencies in intestinal C-type lectins and antimicrobial peptides, which leads to dysbiosis of the intestinal microbiota, alters the mucosal barrier, and promotes development of inflammatory bowel diseases. We investigated whether transgenic (TG) expression of the human regenerating family member 3 alpha gene (REG3A) alters the fecal microbiota and affects development of colitis in mice. METHODS: We performed studies with C57BL/6 mice that express human regenerating family member 3 alpha (hREG3A) in hepatocytes, via the albumin gene promoter. In these mice, hREG3A travels via the bile to the intestinal lumen. Some mice were given dextran sodium sulfate (DSS) to induce colitis. Feces were collected from mice and the composition of the microbiota was analyzed by 16S ribosomal RNA sequencing. The fecal microbiome was also analyzed from mice that express only 1 copy of human REG3A transgene but were fed feces from control mice (not expressing hREG3A) as newborns. Mice expressing hREG3A were monitored for DSS-induced colitis after cohousing or feeding feces from control mice. Colitis was induced in another set of control and hREG3A-TG mice by administration of trinitrobenzene sulfonic acid; some mice were given intrarectal injections of the hREG3A protein. Colon tissues were collected from mice and analyzed by histology and immunohistochemistry to detect mucin 2, as well as by 16S ribosomal RNA fluorescence in situ hybridization, transcriptional analyses, and quantitative polymerase chain reaction. We measured levels of reactive oxygen species (ROS) in bacterial cultures and fecal microbiota using 2',7'-dichlorofluorescein diacetate and flow cytometry. RESULTS: The fecal microbiota of mice that express hREG3A had a significant shift in composition, compared with control mice, with enrichment of Clostridiales (Ruminococcaceae, Lachnospiraceae) and depletion of Bacteroidetes (Prevotellaceae); the TG mice developed less-severe colitis following administration of DSS than control mice, associated with preserved gut barrier integrity and reduced bacterial translocation, epithelial inflammation, and oxidative damage. A similar shift in the composition of the fecal microbiota occurred after a few months in TG mice heterozygous for REG3A that harbored a wild-type maternal microbiota at birth; these mice developed less-severe forms of colitis following DSS administration. Cohoused and germ-free mice fed feces from REG3A-TG mice and given DSS developed less-severe forms of colitis and had reduced lipopolysaccharide activation of the toll-like receptor 4 and increased survival times compared with mice not fed feces from REG3A-TG mice. REG3A TG mice developed only mild colonic inflammation after exposure to 2,4,6-trinitrobenzene sulfonic acid, compared with control mice. Control mice given intrarectal hREG3A and exposed to 2,4,6-trinitrobenzene sulfonic acid showed less colon damage and inflammation than mice not given intrarectal hREG3A. Fecal samples from REG3A-TG mice had lower levels of ROS than feces from control mice during DSS administration. Addition of hREG3A to bacterial cultures reduced levels of ROS and increased survival of oxygen-sensitive commensal bacteria (Faecalibacterium prausnitzii and Roseburia intestinalis). CONCLUSIONS: Mice with hepatocytes that express hREG3A, which travels to the intestinal lumen, are less sensitive to colitis than control mice. We found hREG3A to alter the colonic microbiota by decreasing levels of ROS. Fecal microbiota from REG3A-TG mice protect non-TG mice from induction of colitis. These findings indicate a role for reduction of oxidative stress in preserving the gut microbiota and its ability to prevent inflammation.

Diagnostic preparedness for infectious disease outbreaks.

Diagnostics are crucial in mitigating the effect of disease outbreaks. Because diagnostic development and validation are time consuming, they should be carried out in anticipation of epidemics rather than in response to them. The diagnostic response to the 2014-15 Ebola epidemic, although ultimately effective, was slow and expensive. If a focused mechanism had existed with the technical and financial resources to drive its development ahead of the outbreak, point-of-care Ebola tests supporting a less costly and more mobile response could have been available early on in the diagnosis process. A new partnering model could drive rapid development of tests and surveillance strategies for novel pathogens that emerge in future outbreaks. We look at lessons learned from the Ebola outbreak and propose specific solutions to improve the speed of new assay development and ensure their effective deployment.

Natural variations of copper and sulfur stable isotopes in blood of hepatocellular carcinoma patients.

The widespread hypoxic conditions of the tumor microenvironment can impair the metabolism of bioessential elements such as copper and sulfur, notably by changing their redox state and, as a consequence, their ability to bind specific molecules. Because competing redox state is known to drive isotopic fractionation, we have used here the stable isotope compositions of copper ((65)Cu/(63)Cu) and sulfur ((34)S/(32)S) in the blood of patients with hepatocellular carcinoma (HCC) as a tool to explore the cancer-driven copper and sulfur imbalances. We report that copper is (63)Cu-enriched by ∼0.4‰ and sulfur is (32)S-enriched by ∼1.5‰ in the blood of patients compared with that of control subjects. As expected, HCC patients have more copper in red blood cells and serum compared with control subjects. However, the isotopic signature of this blood extra copper burden is not in favor of a dietary origin but rather suggests a reallocation in the body of copper bound to cysteine-rich proteins such as metallothioneins. The magnitude of the sulfur isotope effect is similar in red blood cells and serum of HCC patients, implying that sulfur fractionation is systemic. The (32)S-enrichment of sulfur in the blood of HCC patients is compatible with the notion that sulfur partly originates from tumor-derived sulfides. The measurement of natural variations of stable isotope compositions, using techniques developed in the field of Earth sciences, can provide new means to detect and quantify cancer metabolic changes and provide insights into underlying mechanisms.

Enterotypes of the human gut microbiome.

Our knowledge of species and functional composition of the human gut microbiome is rapidly increasing, but it is still based on very few cohorts and little is known about variation across the world. By combining 22 newly sequenced faecal metagenomes of individuals from four countries with previously published data sets, here we identify three robust clusters (referred to as enterotypes hereafter) that are not nation or continent specific. We also confirmed the enterotypes in two published, larger cohorts, indicating that intestinal microbiota variation is generally stratified, not continuous. This indicates further the existence of a limited number of well-balanced host-microbial symbiotic states that might respond differently to diet and drug intake. The enterotypes are mostly driven by species composition, but abundant molecular functions are not necessarily provided by abundant species, highlighting the importance of a functional analysis to understand microbial communities. Although individual host properties such as body mass index, age, or gender cannot explain the observed enterotypes, data-driven marker genes or functional modules can be identified for each of these host properties. For example, twelve genes significantly correlate with age and three functional modules with the body mass index, hinting at a diagnostic potential of microbial markers.

HCV core-mediated activation of latent TGF-ß via thrombospondin drives the crosstalk between hepatocytes and stromal environment.

BACKGROUND & AIMS: The mechanisms by which fibrosis, cirrhosis, and hepatocellular carcinoma (HCC) develop during chronic hepatitis C virus (HCV) infection are not fully understood. We previously observed that HCV core protein induced a TGF-ß-dependent epithelial mesenchymal transition, a process contributing to the promotion of cell invasion and metastasis by impacting TGF-ß1 signalling. Here we investigated HCV core capacity to drive increased expression of the active form of TGF-ß1n transgenic mice and hepatoma cell lines. METHODS: We used an in vivo model of HCV core expressing transgenic mice. RESULTS: We observed that about 50% of genes deregulated by core protein expression were TGF-ß1 target genes. Active TGF-ß levels were increased in HCV core transgenic mouse livers. Overexpression of core protein in hepatoma cells increased active TGF-ß levels in culture supernatants and induced Smad2/3 phosphorylation, thus reflecting activation of the TGF-ß signaling pathway. Moreover, our data showed the implication of thrombospondin-1 in core-dependent TGF-ß activation. Finally, hepatoma cells expressing HCV core could activate stellate cells in co-culture and this activation was TGF-ß dependent. CONCLUSIONS: Collectively, these data delineate a novel paradigm where HCV may be related to liver pathogenesis through its ability to induce a local, intrahepatic TGF-ß activation. They argue for a dual impact of HCV core on liver fibrosis and liver carcinogenesis: HCV core could act both as autocrine and paracrine factor modulating TGF-ß responses within hepatocytes and in stromal environment through TGF-ß activation.

Genome-wide association study identifies variants associated with progression of liver fibrosis from HCV infection.

BACKGROUND & AIMS: Polymorphisms in IL28B were shown to affect clearance of hepatitis C virus (HCV) infection in genome-wide association (GWA) studies. Only a fraction of patients with chronic HCV infection develop liver fibrosis, a process that might also be affected by genetic factors. We performed a 2-stage GWA study of liver fibrosis progression related to HCV infection. METHODS: We studied well-characterized HCV-infected patients of European descent who underwent liver biopsies before treatment. We defined various liver fibrosis phenotypes on the basis of METAVIR scores, with and without taking the duration of HCV infection into account. Our GWA analyses were conducted on a filtered primary cohort of 1161 patients using 780,650 single nucleotide polymorphisms (SNPs). We genotyped 96 SNPs with P values <5 × 10(-5) from an independent replication cohort of 962 patients. We then assessed the most interesting replicated SNPs using DNA samples collected from 219 patients who participated in separate GWA studies of HCV clearance. RESULTS: In the combined cohort of 2342 HCV-infected patients, the SNPs rs16851720 (in the total sample) and rs4374383 (in patients who received blood transfusions) were associated with fibrosis progression (P(combined) = 8.9 × 10(-9) and 1.1 × 10(-9), respectively). The SNP rs16851720 is located within RNF7, which encodes an antioxidant that protects against apoptosis. The SNP rs4374383, together with another replicated SNP, rs9380516 (P(combined) = 5.4 × 10(-7)), were linked to the functionally related genes MERTK and TULP1, which encode factors involved in phagocytosis of apoptotic cells by macrophages. CONCLUSIONS: Our GWA study identified several susceptibility loci for HCV-induced liver fibrosis; these were linked to genes that regulate apoptosis. Apoptotic control might therefore be involved in liver fibrosis.

Long COVID: a review and proposed visualization of the complexity of long COVID.

Post-Acute Sequelae of Severe Acute Respiratory Syndrome Coronavirus - 2 (SARS-CoV-2) infection, or Long COVID, is a prevailing second pandemic with nearly 100 million affected individuals globally and counting. We propose a visual description of the complexity of Long COVID and its pathogenesis that can be used by researchers, clinicians, and public health officials to guide the global effort toward an improved understanding of Long COVID and the eventual mechanism-based provision of care to afflicted patients. The proposed visualization or framework for Long COVID should be an evidence-based, dynamic, modular, and systems-level approach to the condition. Furthermore, with further research such a framework could establish the strength of the relationships between pre-existing conditions (or risk factors), biological mechanisms, and resulting clinical phenotypes and outcomes of Long COVID. Notwithstanding the significant contribution that disparities in access to care and social determinants of health have on outcomes and disease course of long COVID, our model focuses primarily on biological mechanisms. Accordingly, the proposed visualization sets out to guide scientific, clinical, and public health efforts to better understand and abrogate the health burden imposed by long COVID.

Biological mechanisms underpinning the development of long COVID.

As COVID-19 evolves from a pandemic to an endemic disease, the already staggering number of people that have been or will be infected with SARS-CoV-2 is only destined to increase, and the majority of humanity will be infected. It is well understood that COVID-19, like many other viral infections, leaves a significant fraction of the infected with prolonged consequences. Continued high number of SARS-CoV-2 infections, viral evolution with escape from post-infection and vaccinal immunity, and reinfections heighten the potential impact of Long COVID. Hence, the impact of COVID-19 on human health will be seen for years to come until more effective vaccines and pharmaceutical treatments become available. To that effect, it is imperative that the mechanisms underlying the clinical manifestations of Long COVID be elucidated. In this article, we provide an in-depth analysis of the evidence on several potential mechanisms of Long COVID and discuss their relevance to its pathogenesis.

Antimicrobial protein REG3A regulates glucose homeostasis and insulin resistance in obese diabetic mice.

Innate immune mediators of pathogen clearance, including the secreted C-type lectins REG3 of the antimicrobial peptide (AMP) family, are known to be involved in the regulation of tissue repair and homeostasis. Their role in metabolic homeostasis remains unknown. Here we show that an increase in human REG3A improves glucose and lipid homeostasis in nutritional and genetic mouse models of obesity and type 2 diabetes. Mice overexpressing REG3A in the liver show improved glucose homeostasis, which is reflected in better insulin sensitivity in normal weight and obese states. Delivery of recombinant REG3A protein to leptin-deficient ob/ob mice or wild-type mice on a high-fat diet also improves glucose homeostasis. This is accompanied by reduced oxidative protein damage, increased AMPK phosphorylation and insulin-stimulated glucose uptake in skeletal muscle tissue. Oxidative damage in differentiated C2C12 myotubes is greatly attenuated by REG3A, as is the increase in gp130-mediated AMPK activation. In contrast, Akt-mediated insulin action, which is impaired by oxidative stress, is not restored by REG3A. These data highlight the importance of REG3A in controlling oxidative protein damage involved in energy and metabolic pathways during obesity and diabetes, and provide additional insight into the dual function of host-immune defense and metabolic regulation for AMP.

Monkeypox (Mpox) requires continued surveillance, vaccines, therapeutics and mitigating strategies.

The widespread outbreak of the monkeypox virus (MPXV) recognized in 2022 poses new challenges for public healthcare systems worldwide. With more than 86,000 people infected, there is concern that MPXV may become endemic outside of its original geographical area leading to repeated human spillover infections or continue to be spread person-to-person. Fortunately, classical public health measures (e.g., isolation, contact tracing and quarantine) and vaccination have blunted the spread of the virus, but cases are continuing to be reported in 28 countries in March 2023. We describe here the vaccines and drugs available for the prevention and treatment of MPXV infections. However, although their efficacy against monkeypox (mpox) has been established in animal models, little is known about their efficacy in the current outbreak setting. The continuing opportunity for transmission raises concerns about the potential for evolution of the virus and for expansion beyond the current risk groups. The priorities for action are clear: 1) more data on the efficacy of vaccines and drugs in infected humans must be gathered; 2) global collaborations are necessary to ensure that government authorities work with the private sector in developed and low and middle income countries (LMICs) to provide the availability of treatments and vaccines, especially in historically endemic/enzootic areas; 3) diagnostic and surveillance capacity must be increased to identify areas and populations where the virus is present and may seed resurgence; 4) those at high risk of severe outcomes (e.g., immunocompromised, untreated HIV, pregnant women, and inflammatory skin conditions) must be informed of the risk of infection and be protected from community transmission of MPXV; 5) engagement with the hardest hit communities in a non-stigmatizing way is needed to increase the understanding and acceptance of public health measures; and 6) repositories of monkeypox clinical samples, including blood, fluids, tissues and lesion material must be established for researchers. This MPXV outbreak is a warning that pandemic preparedness plans need additional coordination and resources. We must prepare for continuing transmission, resurgence, and repeated spillovers of MPXV.

Human hepatocarcinoma-intestine-pancreas/pancreatitis-associated protein cures fas-induced acute liver failure in mice by attenuating free-radical damage in injured livers.

UNLABELLED: Acute liver failure (ALF) is a rare syndrome with a difficult clinical management and a high mortality rate. During ALF, several molecular pathways governing oxidative stress and apoptosis are activated to induce massive tissue injury and suppress cell proliferation. There are few anti-ALF drug candidates, among which is the C-type lectin Reg3α, or human hepatocarcinoma-intestine-pancreas/pancreatitis-associated protein (HIP/PAP), which displayed promising properties for tissue regeneration and protection against cellular stress in transgenic mice. We report on substantial preclinical and clinical advances in the development of a recombinant (rc) full-length human HIP/PAP protein as an anti-ALF drug. The curative effects and mechanisms of action of rcHIP/PAP were investigated in murine Fas-induced ALF. Primary hepatocytes were cultured with cytotoxic doses of tumor necrosis factor α/actinomycin-D, transforming growth factor ß, agonistic Fas antibody or hydrogen peroxide, and various concentrations of rcHIP/PAP. Cell viability, proliferation index, apoptosis, and oxidation were monitored. We found that rcHIP/PAP significantly improved survival in Fas-intoxicated mice in a dose-dependent and time-dependent manner, with optimum effects when it was injected at advanced stages of ALF. Primary hepatocytes were efficiently protected against multiple cell death signals by rcHIP/PAP. This survival benefit was linked to a depletion of oxidized biomolecules in injured liver cells due to a strong reactive oxygen species scavenging activity of rcHIP/PAP. Clinically, an escalating dose phase 1 trial demonstrated a good tolerability and pharmacokinetic profile of rcHIP/PAP in healthy subjects. CONCLUSION: The rcHIP/PAP protein exhibited significant curative properties against ALF in mice. It is a free-radical scavenger that targets a broad spectrum of death effectors and favors liver regeneration. The good safety profile of rcHIP/PAP during a phase 1 trial encourages evaluation of its efficacy in patients with ALF.

Identification of cellular targets in human intrahepatic cholangiocarcinoma using laser microdissection and accurate mass and time tag proteomics.

Obtaining accurate protein profiles from homogeneous cell populations in heterogeneous tissues can enhance the capability to discover protein biomarkers. In this context, methodologies to access specific cellular populations and analyze their proteome with exquisite sensitivity have to be selected. We report here the results of an investigation using a combination of laser microdissection and accurate mass and time tag proteomics. The study was aimed at the precise determination of proteome alterations in intrahepatic cholangiocarcinoma ICC, a markedly heterogeneous tumor. This cancer, which is difficult to diagnose and carries a very poor prognosis, has shown an unexplained increase in incidence over the last few years. Among a pool of 574 identified proteins, we were able to report on altered abundance patterns affecting 39 proteins conforming to a variety of potential tumorigenic pathways. The reliability of the proteomics results was confirmed by Western blot and immunohistochemistry on matched samples. Most of the proteins displaying perturbed abundances had not yet been described in the setting of ICC. These include proteins involved in cell mobility and actin cytoskeleton remodeling, which may participate in the epithelial to mesenchymal transition, a process invoked in migration and invasion of cancer cells. The biological relevance of these findings was explored using a tissue microarray. An increased abundance of vimentin was thus detected in 70% of ICC and none of the controls. These results suggest that vimentin could play a role in the aggressiveness of ICC and provide a basis for the serious outcome of this cancer.

A randomized double-blind placebo-controlled clinical trial of nitazoxanide for treatment of mild or moderate COVID-19.

BACKGROUND: There is an urgent need for treatments of mild or moderate COVID-19 in an outpatient setting. METHODS: A randomized double-blind placebo-controlled clinical trial in 36 centers in the U.S. between August 2020 and February 2021 investigated the safety and effectiveness of oral nitazoxanide 600 mg twice daily for five days in outpatients with symptoms of mild or moderate COVID-19 enrolled within 72 h of symptom onset (ClinicalTrials.gov NCT04486313). Efficacy endpoints were time to sustained clinical recovery (TSR, a novel primary endpoint) and proportion of participants progressing to severe illness within 28 days (key secondary). FINDINGS: 1092 participants were enrolled. 379 with laboratory-confirmed SARS-CoV-2 infection were analyzed. In the primary analysis, median (IQR) TSR were 13·3 (6·3, >21) and 12·4 (7·2, >21) days for the nitazoxanide and placebo groups, respectively (p = 0·88). 1 of 184 (0·5%) treated with nitazoxanide progressed to severe illness compared to 7 of 195 (3·6%) treated with placebo (key secondary analysis, odds ratio 5·6 [95% CI 0·7 - 46·1], relative risk reduction 85%, p = 0·07). In the pre-defined stratum with mild illness at baseline, nitazoxanide-treated participants experienced reductions in median TSR (3·1 days, p = 0·09) and usual health (5·2 days, p < 0·01) compared to placebo. Nitazoxanide was safe and well tolerated. INTERPRETATION: Further trials with larger numbers are warranted to evaluate efficacy of nitazoxanide therapy in preventing progression to severe illness in patients at high risk of severe illness and reducing TSR in patients with mild illness.

Sorafenib and nitazoxanide disrupt mitochondrial function and inhibit regrowth capacity in three-dimensional models of hepatocellular and colorectal carcinoma.

Quiescent cancer cells in malignant tumors can withstand cell-cycle active treatment and cause cancer spread and recurrence. Three-dimensional (3D) cancer cell models have led to the identification of oxidative phosphorylation (OXPHOS) as a context-dependent vulnerability. The limited treatment options for advanced hepatocellular carcinoma (HCC) and colorectal carcinoma (CRC) metastatic to the liver include the multikinase inhibitors sorafenib and regorafenib. Off-target effects of sorafenib and regorafenib are related to OXPHOS inhibition; however the importance of this feature to the effect on tumor cells has not been investigated in 3D models. We began by assessing global transcriptional responses in monolayer cell cultures, then moved on to multicellular tumor spheroids (MCTS) and tumoroids generated from a CRC patient. Cells were treated with chemotherapeutics, kinase inhibitors, and the OXPHOS inhibitors. Cells grown in 3D cultures were sensitive to the OXPHOS inhibitor nitazoxanide, sorafenib, and regorafenib and resistant to other multikinase inhibitors and chemotherapeutic drugs. Furthermore, nitazoxanide and sorafenib reduced viability, regrowth potential and inhibited mitochondrial membrane potential in an additive manner at clinically relevant concentrations. This study demonstrates that the OXPHOS inhibition caused by sorafenib and regorafenib parallels 3D activity and can be further investigated for new combination strategies.