Microdissected tumor cuboids: a microscale cancer model that retains a complex tumor microenvironment.

Present cancer disease models - typically based on cell cultures and animal models that lack the human tumor microenvironment (TME) - are extremely poor predictors of human disease outcomes. Microscale cancer models that combine the micromanipulation of tissues and fluids offer the exciting possibility of miniaturizing the drug testing workflow, enabling inexpensive, more efficient tests of high clinical biomimicry that maximize the use of scarce human tissue and minimize animal testing. Critically, these microscale models allow for precisely addressing the impact of the structural features of the heterogeneous TME to properly target and understand the contributions of these unique zones to therapeutic response. We have recently developed a precision slicing method that yields large numbers of cuboidal micro-tissues ("cuboids", ∼ (400 µm) 3 ) from a single tumor biopsy. Here we evaluate cuboids from syngeneic mouse tumor models and human tumors, which contain native immune cells, as models for drug and immunotherapy evaluation. We characterize relevant TME parameters, such as their cellular architecture (immune cells and vasculature), cytokine secretion, proteomics profiles, and their response to drug panels in multi-well arrays. Despite the cutting procedure and the time spent in culture (up to 7 days), the cuboids display strong functional responses such as cytokine and drug responses. Overall, our results suggest that cuboids make an excellent model for applications that require the TME, such as immunotherapy drug evaluations, including for clinical trials and personalized oncology approaches.

Label-Free, Real-Time Monitoring of Cytochrome C Responses to Drugs in Microdissected Tumor Biopsies with a Multi-Well Aptasensor Platform.

Functional assays on intact tumor biopsies can potentially complement and extend genomics-based approaches for precision oncology, drug testing, and organs-on-chips cancer disease models by capturing key determinants of therapeutic response, such as tissue architecture, tumor heterogeneity, and the tumor microenvironment. Currently, most of these assays rely on fluorescent labeling, a semi-quantitative method best suited to be a single-time-point terminal assay or labor-intensive terminal immunostaining analysis. Here, we report integrated aptamer electrochemical sensors for on-chip, real-time monitoring of increases of cytochrome C, a cell death indicator, from intact microdissected tissues with high affinity and specificity. The platform features a multi-well sensor layout and a multiplexed electronic setup. The aptasensors measure increases in cytochrome C in the supernatant of mouse or human microdissected tumors after exposure to various drug treatments. Since the aptamer probe can be easily exchanged to recognize different targets, the platform could be adapted for multiplexed monitoring of various biomarkers, providing critical information on the tumor and its microenvironment. This approach could not only help develop more advanced cancer disease models but also apply to other complex in vitro disease models, such as organs-on-chips and organoids.

Recruitment of BAG2 to DNAJ-PKAc scaffolds promotes cell survival and resistance to drug-induced apoptosis in fibrolamellar carcinoma.

The DNAJ-PKAc fusion kinase is a defining feature of fibrolamellar carcinoma (FLC). FLC tumors are notoriously resistant to standard chemotherapies, with aberrant kinase activity assumed to be a contributing factor. By combining proximity proteomics, biochemical analyses, and live-cell photoactivation microscopy, we demonstrate that DNAJ-PKAc is not constrained by A-kinase anchoring proteins. Consequently, the fusion kinase phosphorylates a unique array of substrates, including proteins involved in translation and the anti-apoptotic factor Bcl-2-associated athanogene 2 (BAG2), a co-chaperone recruited to the fusion kinase through association with Hsp70. Tissue samples from patients with FLC exhibit increased levels of BAG2 in primary and metastatic tumors. Furthermore, drug studies implicate the DNAJ-PKAc/Hsp70/BAG2 axis in potentiating chemotherapeutic resistance. We find that the Bcl-2 inhibitor navitoclax enhances sensitivity to etoposide-induced apoptosis in cells expressing DNAJ-PKAc. Thus, our work indicates BAG2 as a marker for advanced FLC and a chemotherapeutic resistance factor in DNAJ-PKAc signaling scaffolds.

Quantification of Hepatocellular Carcinoma Vascular Dynamics With Contrast-Enhanced Ultrasound for LI-RADS Implementation.

OBJECTIVE: The aim of this study is to describe a comprehensive contrast-enhanced ultrasound (CEUS) imaging protocol and analysis method to implement CEUS LI-RADS (Liver Imaging Reporting and Data System) in a quantifiable manner. The methods that are validated with a prospective single-center study aim to simplify CEUS LI-RADS evaluation, remove observer bias, and potentially improve the sensitivity of CEUS LI-RADS. MATERIALS AND METHODS: This prospective single-center study enrolled patients with hepatocellular carcinoma (April 2021-June 2022; N = 31; mean age ± SD, 67 ± 6 years; 24 men/7 women). For each patient, at least 2 CEUS loops spanning over 5 minutes were collected for different lesion scan planes using an articulated arm to hold the transducer. Automatic respiratory gating and motion compensation algorithms removed errors due to breathing motion. The long axis of the lesion was measured in the contrast and fundamental images to capture nodule size. Parametric processing of time-intensity curve analysis on linearized data provided quantifiable information of the wash-in and washout dynamics via rise time ( RT ) and degree of washout ( DW ) parameters extracted from the time-intensity curve, respectively. A Welch t test was performed between lesion and parenchyma RT for each lesion to confirm statistically significant differences. P values for bootstrapped 95% confidence intervals of the relative degree of washout ( rDW ), ratio of DW between the lesion and surrounding parenchyma, were computed to quantify lesion washout. Coefficient of variation (COV) of RT , DW , and rDW was calculated for each patient between injections for both the lesion and surrounding parenchyma to gauge reproducibility of these metrics. Spearman rank correlation tests were performed among size, RT , DW , and rDW values to evaluate statistical dependence between the variables. RESULTS: The mean ± SD lesion diameter was 23 ± 8 mm. The RT for all lesions, capturing arterial phase hyperenhancement, was shorter than that of surrounding liver parenchyma ( P < 0.05). All lesions also demonstrated significant ( P < 0.05) but variable levels of washout at both 2-minute and 5-minute time points, quantified in rDW . The COV of RT for the lesion and surrounding parenchyma were both 11%, and the COV of DW and rDW at 2 and 5 minutes ranged from 22% to 31%. Statistically significant relationships between lesion and parenchyma RT and between lesion RT and lesion DW at the 2- and 5-minute time points were found ( P < 0.05). CONCLUSIONS: The imaging protocol and analysis method presented provide robust, quantitative metrics that describe the dynamic vascular patterns of LI-RADS 5 lesions classified as hepatocellular carcinomas. The RT of the bolus transit quantifies the arterial phase hyperenhancement, and the DW and rDW parameters quantify the washout from linearized CEUS intensity data. This unique methodology is able to implement the CEUS-LIRADS scheme in a quantifiable manner for the first time and remove its existing issues of currently being qualitative and suffering from subjective evaluations.

Organotypic Models for Functional Drug Testing of Human Cancers.

In the era of personalized oncology, there have been accelerated efforts to develop clinically relevant platforms to test drug sensitivities of individual cancers. An ideal assay will serve as a diagnostic companion to inform the oncologist of the various treatments that are sensitive and insensitive, thus improving outcome while minimizing unnecessary toxicities and costs. To date, no such platform exists for clinical use, but promising approaches are on the horizon that take advantage of improved techniques in creating human cancer models that encompass the entire tumor microenvironment, alongside technologies for assessing and analyzing tumor response. This review summarizes a number of current strategies that make use of intact human cancer tissues as organotypic cultures in drug sensitivity testing.

Multiple Linear-Combination Security Network Coding.

In this paper, we put forward the model of multiple linear-combination security multicast network coding, where the wiretapper desires to obtain some information about a predefined set of multiple linear combinations of the source symbols by eavesdropping any one (but not more than one) channel subset up to a certain size r, referred to as the security level. For this model, the security capacity is defined as the maximum average number of source symbols that can be securely multicast to all sink nodes for one use of the network under the linear-combination security constraint. For any security level and any linear-combination security constraint, we fully characterize the security capacity in terms of the ratio of the rank of the linear-combination security constraint to the number of source symbols. Also, we develop a general construction of linear security network codes. Finally, we investigate the asymptotic behavior of the security capacity for a sequence of linear-combination security models and discuss the asymptotic optimality of our code construction.

Recruitment of BAG2 to DNAJ-PKAc scaffolds promotes cell survival and resistance to drug-induced apoptosis in fibrolamellar carcinoma.

The DNAJ-PKAc fusion kinase is a defining feature of the adolescent liver cancer fibrolamellar carcinoma (FLC). A single lesion on chromosome 19 generates this mutant kinase by creating a fused gene encoding the chaperonin binding domain of Hsp40 (DNAJ) in frame with the catalytic core of protein kinase A (PKAc). FLC tumors are notoriously resistant to standard chemotherapies. Aberrant kinase activity is assumed to be a contributing factor. Yet recruitment of binding partners, such as the chaperone Hsp70, implies that the scaffolding function of DNAJ- PKAc may also underlie pathogenesis. By combining proximity proteomics with biochemical analyses and photoactivation live-cell imaging we demonstrate that DNAJ-PKAc is not constrained by A-kinase anchoring proteins. Consequently, the fusion kinase phosphorylates a unique array of substrates. One validated DNAJ-PKAc target is the Bcl-2 associated athanogene 2 (BAG2), a co-chaperone recruited to the fusion kinase through association with Hsp70. Immunoblot and immunohistochemical analyses of FLC patient samples correlate increased levels of BAG2 with advanced disease and metastatic recurrences. BAG2 is linked to Bcl-2, an anti-apoptotic factor that delays cell death. Pharmacological approaches tested if the DNAJ- PKAc/Hsp70/BAG2 axis contributes to chemotherapeutic resistance in AML12 DNAJ-PKAc hepatocyte cell lines using the DNA damaging agent etoposide and the Bcl-2 inhibitor navitoclax. Wildtype AML12 cells were susceptible to each drug alone and in combination. In contrast, AML12 DNAJ-PKAc cells were moderately affected by etoposide, resistant to navitoclax, but markedly susceptible to the drug combination. These studies implicate BAG2 as a biomarker for advanced FLC and a chemotherapeutic resistance factor in DNAJ-PKAc signaling scaffolds.

Republication of "Percutaneous Basal Closing Wedge Osteotomy of the First Metatarsal in the Treatment of Moderate to Severe Hallux Valgus and Its Short-Term Clinical Outcomes".

Background: Although percutaneous surgery for the treatment of hallux valgus is popular in Europe, there is sparse English written literature documenting its efficacy. This study described the operative techniques using percutaneous basal closing wedge osteotomy of the first metatarsal in correction of moderate to severe hallux valgus (HV) and its short-term clinical outcomes. We postulated that satisfactory correction of hallux valgus (HV) angle, intermetatarsal (IM) angle, and patients' clinical outcomes could be achieved with this technique. Methods: We conducted a retrospective review of 25 feet in 23 patients who underwent a percutaneous basal closing wedge osteotomy of the first metatarsal (MT1) combined with a mini-open modified McBride procedure and mini-open resection of medial eminence. Follow-up averaged 21.5 months. Radiographic outcomes included pre- and postoperative HV angle, IM angle, absolute and relative shortening of MT1, and time to union. American Orthopaedic Foot & Ankle Society (AOFAS) scores were compared between pre- and postoperatively. Results: The average HV angle improved from 39.4 (range, 29-58.3) degrees preoperatively to 14.7 (range, 0.1-23.2) degrees postoperatively (P < .05). IM angle improved from 14.9 (range, 6.7-22.4) degrees to 6.6 (range, 0.9-14.8) degrees (P < .05). The average absolute shortening was 3.8 (range, 0.27-12.91) mm and the relative shortening was 0.8 (range, 0.05-1.91) mm. There was no delayed union or malunion at the osteotomy site. The average AOFAS score improved from 39 (range, 12-50) to 81 (range, 70-93) (P < .05). Conclusions: Satisfactory hallux valgus deformity correction and patients' outcomes were achieved with this technique. Our results are similar to results reported in other studies using open techniques. There was no malunion or delayed union of the osteotomy. Level of Evidence: Level IV, case series study.

Oncogenic PKA signaling increases c-MYC protein expression through multiple targetable mechanisms.

Genetic alterations that activate protein kinase A (PKA) are found in many tumor types. Yet, their downstream oncogenic signaling mechanisms are poorly understood. We used global phosphoproteomics and kinase activity profiling to map conserved signaling outputs driven by a range of genetic changes that activate PKA in human cancer. Two signaling networks were identified downstream of PKA: RAS/MAPK components and an Aurora Kinase A (AURKA)/glycogen synthase kinase (GSK3) sub-network with activity toward MYC oncoproteins. Findings were validated in two PKA-dependent cancer models: a novel, patient-derived fibrolamellar carcinoma (FLC) line that expresses a DNAJ-PKAc fusion and a PKA-addicted melanoma model with a mutant type I PKA regulatory subunit. We identify PKA signals that can influence both de novo translation and stability of the proto-oncogene c-MYC. However, the primary mechanism of PKA effects on MYC in our cell models was translation and could be blocked with the eIF4A inhibitor zotatifin. This compound dramatically reduced c-MYC expression and inhibited FLC cell line growth in vitro. Thus, targeting PKA effects on translation is a potential treatment strategy for FLC and other PKA-driven cancers.

Blockade of interleukin 10 potentiates antitumour immune function in human colorectal cancer liver metastases.

OBJECTIVE: Programmed cell death protein 1 (PD-1) checkpoint inhibition and adoptive cellular therapy have had limited success in patients with microsatellite stable colorectal cancer liver metastases (CRLM). We sought to evaluate the effect of interleukin 10 (IL-10) blockade on endogenous T cell and chimeric antigen receptor T (CAR-T) cell antitumour function in CRLM slice cultures. DESIGN: We created organotypic slice cultures from human CRLM (n=38 patients' tumours) and tested the antitumour effects of a neutralising antibody against IL-10 (αIL-10) both alone as treatment and in combination with exogenously administered carcinoembryonic antigen (CEA)-specific CAR-T cells. We evaluated slice cultures with single and multiplex immunohistochemistry, in situ hybridisation, single-cell RNA sequencing, reverse-phase protein arrays and time-lapse fluorescent microscopy. RESULTS: αIL-10 generated a 1.8-fold increase in T cell-mediated carcinoma cell death in human CRLM slice cultures. αIL-10 significantly increased proportions of CD8+ T cells without exhaustion transcription changes, and increased human leukocyte antigen - DR isotype (HLA-DR) expression of macrophages. The antitumour effects of αIL-10 were reversed by major histocompatibility complex class I or II (MHC-I or MHC-II) blockade, confirming the essential role of antigen presenting cells. Interrupting IL-10 signalling also rescued murine CAR-T cell proliferation and cytotoxicity from myeloid cell-mediated immunosuppression. In human CRLM slices, αIL-10 increased CEA-specific CAR-T cell activation and CAR-T cell-mediated cytotoxicity, with nearly 70% carcinoma cell apoptosis across multiple human tumours. Pretreatment with an IL-10 receptor blocking antibody also potentiated CAR-T function. CONCLUSION: Neutralising the effects of IL-10 in human CRLM has therapeutic potential as a stand-alone treatment and to augment the function of adoptively transferred CAR-T cells.

Response of Human Liver Tissue to Innate Immune Stimuli.

Precision-cut human liver slice cultures (PCLS) have become an important alternative immunological platform in preclinical testing. To further evaluate the capacity of PCLS, we investigated the innate immune response to TLR3 agonist (poly-I:C) and TLR4 agonist (LPS) using normal and diseased liver tissue. Pathological liver tissue was obtained from patients with active chronic HCV infection, and patients with former chronic HCV infection cured by recent Direct-Acting Antiviral (DAA) drug therapy. We found that hepatic innate immunity in response to TLR3 and TLR4 agonists was not suppressed but enhanced in the HCV-infected tissue, compared with the healthy controls. Furthermore, despite recent HCV elimination, DAA-cured liver tissue manifested ongoing abnormalities in liver immunity: sustained abnormal immune gene expression in DAA-cured samples was identified in direct ex vivo measurements and in TLR3 and TLR4 stimulation assays. Genes that were up-regulated in chronic HCV-infected liver tissue were mostly characteristic of the non-parenchymal cell compartment. These results demonstrated the utility of PCLS in studying both liver pathology and innate immunity.

Glypican-3-Targeted 227Th α-Therapy Reduces Tumor Burden in an Orthotopic Xenograft Murine Model of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a significant cause of morbidity and mortality worldwide, with limited therapeutic options for advanced disease. Targeted α-therapy is an emerging class of targeted cancer therapy in which α-particle-emitting radionuclides, such as 227Th, are delivered specifically to cancer tissue. Glypican-3 (GPC3) is a cell surface glycoprotein highly expressed on HCC. In this study, we describe the development and in vivo efficacy of a 227Th-labeled GPC3-targeting antibody conjugate (227Th-octapa-αGPC3) for treatment of HCC in an orthotopic murine model. Methods: The chelator p-SCN-Bn-H4octapa-NCS (octapa) was conjugated to a GPC3-targeting antibody (αGPC3) for subsequent 227Th radiolabeling (octapa-αGPC3). Conditions were varied to optimize radiolabeling of 227Th. In vitro stability was evaluated by measuring the percentage of protein-bound 227Th by γ-ray spectroscopy. An orthotopic athymic Nu/J murine model using HepG2-Red-FLuc cells was developed. Biodistribution and blood clearance of 227Th-octapa-αGPC3 were evaluated in tumor-bearing mice. The efficacy of 227Th-octapa-αGPC3 was assessed in tumor-bearing animals with serial measurement of serum α-fetoprotein at 23 d after injection. Results: Octapa-conjugated αGPC3 provided up to 70% 227Th labeling yield in 2 h at room temperature. In the presence of ascorbate, at least 97.8% of 227Th was bound to αGPC3-octapa after 14 d in phosphate-buffered saline. In HepG2-Red-FLuc tumor-bearing mice, highly specific GPC3 targeting was observed, with significant 227Th-octapa-αGPC3 accumulation in the tumor over time and minimal accumulation in normal tissue. Twenty-three days after treatment, a significant reduction in tumor burden was observed in mice receiving a 500 kBq/kg dose of 227Th-octapa-αGPC3 by tail-vein injection. No acute off-target toxicity was observed, and no animals died before termination of the study. Conclusion:227Th-octapa-αGPC3 was observed to be stable in vitro; maintain high specificity for GPC3, with favorable biodistribution in vivo; and result in significant antitumor activity without significant acute off-target toxicity in an orthotopic murine model of HCC.

IWATE criteria are associated with perioperative outcomes in robotic hepatectomy: a retrospective review of 225 resections.

BACKGROUND: Robotic hepatectomy (RH) is increasingly utilized for minor and major liver resections. The IWATE criteria were developed to classify minimally invasive liver resections by difficulty. The objective of this study was to apply the IWATE criteria in RH and to describe perioperative and oncologic outcomes of RH over the last decade at our institution. METHODS: Perioperative and oncologic outcomes of patients who underwent RH between 2011 and 2019 were retrospectively collected. The difficulty level of each operation was assessed using the IWATE criteria, and outcomes were compared at each level. Univariate linear regression was performed to characterize the relationship between IWATE criteria and perioperative outcomes (OR time, EBL, and LOS), and a multivariable model was also developed to address potential confounding by patient characteristics (age, sex, BMI, prior abdominal surgery, ASA class, and simultaneous non-hepatectomy operation). RESULTS: Two hundred and twenty-five RH were performed. Median IWATE criteria for RH were 6 (IQR 5-9), with low, intermediate, advanced, and expert resections accounting for 23% (n = 51), 34% (n = 77), 32% (n = 72), and 11% (n = 25) of resections, respectively. The majority of resections were parenchymal-sparing approaches, including anatomic segmentectomies and non-anatomic partial resections. 30-day complication rate was 14%, conversion to open surgery occurred in 9 patients (4%), and there were no deaths within 30 days postoperatively. In the univariate linear regression analysis, IWATE criteria were positively associated with OR time, EBL, and LOS. In the multivariable model, IWATE criteria were independently associated with greater OR time, EBL, and LOS. Two-year overall survival for hepatocellular carcinoma and intrahepatic cholangiocarcinoma was 94% and 50%, respectively. CONCLUSION: In conclusion, the IWATE criteria are associated with surgical outcomes after RH. This series highlights the utility of RH for difficult hepatic resections, particularly parenchymal-sparing resections in the posterosuperior sector, extending the indication of minimally invasive hepatectomy in experienced hands and potentially offering select patients an alternative to open hepatectomy or other less definitive liver-directed treatment options.

Safety of Image-Guided Treatment of the Liver with Ultrasound and Microbubbles in an in Vivo Porcine Model.

Ultrasound and microbubbles are useful for both diagnostic imaging and targeted drug delivery, making them ideal conduits for theranostic interventions. Recent reports have indicated the preclinical success of microbubble cavitation for enhancement of chemotherapy in abdominal tumors; however, there have been limited studies and variable efficacy in clinical implementation of this technique. This is likely because in contrast to the high pressures and long cycle lengths seen in successful preclinical work, current clinical implementation of microbubble cavitation for drug delivery generally involves low acoustic pressures and short cycle lengths to fit within clinical guidelines. To translate the preclinical parameter space to clinical adoption, a relevant safety study in a healthy large animal is required. Therefore, the purpose of this work was to evaluate the safety of ultrasound cavitation treatment (USCTx) in a healthy porcine model using a modified Philips EPIQ with S5-1 as the focused source. We performed USCTx on eight healthy pigs and monitored health over the course of 1 wk. We then performed an acute study of USCTx to evaluate immediate tissue damage. Contrast-enhanced ultrasound exams were performed before and after each treatment to investigate perfusion changes within the treated areas, and blood and urine were evaluated for liver damage biomarkers. We illustrate, through quantitative analysis of contrast-enhanced ultrasound data, blood and urine analyses and histology, that this technique and the parameter space considered are safe within the time frame evaluated. With its safety confirmed using a clinical-grade ultrasound scanner and contrast agent, USCTx could be easily translated into clinical trials for improvement of chemotherapy delivery. This represents the first safety study assessing the bio-effects of microbubble cavitation from relevant ultrasound parameters in a large animal model.

Protocol for tissue slice cultures from human solid tumors to study therapeutic response.

The impact of systemic therapy on the tumor microenvironment has been difficult to study in human solid tumors. Our protocol describes steps for establishing slice cultures to investigate response to chemotherapies, immunotherapies, or adoptive cell therapies. Endpoints include changes in viability, histology, live-cell imaging, and multi-omics analyses. The protocol has been applied to a broad array of gastrointestinal malignancies. Culture conditions and treatment parameters can be modified for specific experiments. The platform is highly flexible and easy to manipulate. For complete details on the use and execution of this protocol, please refer to Kenerson et al. (2020), Jabbari et al. (2020), Brempelis et al. (2020), and Jiang et al. (2017).

Hepatic artery aneurysm in the setting of focal nodular hyperplasia.

Hepatic artery aneurysms (HAA) are rare and may be seen in the setting of infection and vascular disease. Clinical presentation is variable but many are found incidentally during imaging studies. The association of HAA with focal nodular hyperplasia (FNH) is rarely reported in literature. We present the case of a 68-year-old woman found to have a hepatic artery aneurysm and hepatic mass, both within the same liver segment. FNH and hepatic adenomas share similar imaging features but have different treatments due to malignant potential of the latter, and biopsy should be performed when adenoma cannot be excluded. In this case biopsy of the mass revealed it to be FNH and the aneurysm was treated with embolization rather than surgery.

Glypican-3 targeted delivery of 89Zr and 90Y as a theranostic radionuclide platform for hepatocellular carcinoma.

Glypican-3 (GPC3) is a tumor associated antigen expressed by hepatocellular carcinoma (HCC) cells. This preclinical study evaluated the efficacy of a theranostic platform using a GPC3-targeting antibody αGPC3 conjugated to zirconium-89 (89Zr) and yttrium-90 (90Y) to identify, treat, and assess treatment response in a murine model of HCC. A murine orthotopic xenograft model of HCC was generated. Animals were injected with 89Zr-labeled αGPC3 and imaged with a small-animal positron emission/computerized tomography (PET/CT) imaging system (immuno-PET) before and 30 days after radioimmunotherapy (RIT) with 90Y-labeled αGPC3. Serum alpha fetoprotein (AFP), a marker of tumor burden, was measured. Gross tumor volume (GTV) and SUVmax by immuno-PET was measured using fixed intensity threshold and manual segmentation methods. Immuno-PET GTV measurements reliably quantified tumor burden prior to RIT, strongly correlating with serum AFP (R2 = 0.90). Serum AFP was significantly lower 30 days after RIT in 90Y-αGPC3 treated animals compared to those untreated (p = 0.01) or treated with non-radiolabeled αGPC3 (p = 0.02). Immuno-PET GTV measurements strongly correlated with tumor burden after RIT (R2 = 0.87), and GTV of animals treated with 90Y-αGPC3 was lower than in animals who did not receive treatment or were treated with non-radiolabeled αGPC3, although this only trended toward statistical significance. A theranostic platform utilizing GPC3 targeted 89Zr and 90Y effectively imaged, treated, and assessed response after radioimmunotherapy in a GPC3-expressing HCC xenograft model.

Functional responses of resident human T cells in intact liver tissue.

The liver contains a rich mix of T cells, including activated T cells, tissue-resident memory T cells and cells undergoing apoptosis. When antigens are presented in this milieu the default result is functional tolerance. T cell tolerance in the liver could be constitutive, or it could be adaptive, in which case liver cells would become unresponsive after encountering antigen in the liver context. To test this model, we evaluated the potential of human liver T cells to respond to T cell receptor ligation in liver tissue slice cultures. These T cells contained an actively motile subset of CD4+ T cells marked by CCR7 and CD62L, and fully functional subsets of CD4+ and CD8+ T cells that synthesized effector cytokines but subsequently assumed an exhausted phenotype. These data favor the model that human liver T cells are not constitutively tolerant but undergo adaptive tolerance after activation.

Altered vitamin A metabolism in human liver slices corresponds to fibrogenesis.

All-trans-retinoic acid (atRA), the active metabolite of vitamin A, has antifibrogenic properties in vitro and in animal models. Liver vitamin A homeostasis is maintained by cell-specific enzymatic activities including storage in hepatic stellate cells (HSCs), secretion into circulation from hepatocytes, and formation and clearance of atRA. During chronic liver injury, HSC activation is associated with a decrease in liver retinyl esters and retinol concentrations. atRA is synthesized through two enzymatic steps from retinol, but it is unknown if the loss of retinoid stores is associated with changes in atRA formation and which cell types contribute to the metabolic changes. The aim of this study was to determine if the vitamin A metabolic flux is perturbed in acute liver injury, and if changes in atRA concentrations are associated with HSC activation and collagen expression. At basal levels, HSC and Kupffer cells expressed key genes involved in vitamin A metabolism, whereas after acute liver injury, complex changes to the metabolic flux were observed in liver slices. These changes include a reproducible spike in atRA tissue concentrations, decreased retinyl ester and atRA formation rate, and time-dependent changes to the expression of metabolizing enzymes. Kinetic simulations suggested that oxidoreductases are important in determining retinoid metabolic flux after liver injury. These early changes precede HSC activation and upregulation of profibrogenic gene expression, which were inversely correlated with atRA tissue concentrations, suggesting that HSC and Kupffer cells are key cells involved in changes to vitamin A metabolic flux and signaling after liver injury. Study Highlights WHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC? Vitamin A is metabolized in the liver for storage as retinyl esters in hepatic stellate cell (HSCs) or to all-trans-retinoic acid (atRA), an active metabolite with antifibrogenic properties. Following chronic liver injury, vitamin A metabolic flux is perturbed, and HSC activation leads to diminished retinoid stores. WHAT QUESTION DID THIS STUDY ADDRESS? Do changes in the expression of vitamin A metabolizing enzymes explain changes in atRA concentrations and the regulation of fibrosis following acute liver injury? WHAT DOES THIS STUDY ADD TO OUR KNOWLEDGE? In healthy liver, both HSC and Kupffer cells may mediate vitamin A homeostasis. Following acute liver injury, complex changes in metabolizing enzyme expression/activity alter the metabolic flux of retinoids, resulting in a transient peak in atRA concentrations. The atRA concentrations are inversely correlated with profibrogenic gene expression, HSC activation, and collagen deposition. HOW MIGHT THIS CHANGE CLINICAL PHARMACOLOGY OR TRANSLATIONAL SCIENCE? Improved understanding of altered vitamin A metabolic flux in acute liver injury may provide insight into cell-specific contributions to vitamin A loss and lead to novel interventions in liver fibrosis.

Modulation of Immune Checkpoints by Chemotherapy in Human Colorectal Liver Metastases.

Metastatic colorectal cancer (CRC) is a major cause of cancer-related death, and incidence is rising in younger populations (younger than 50 years). Current chemotherapies can achieve response rates above 50%, but immunotherapies have limited value for patients with microsatellite-stable (MSS) cancers. The present study investigates the impact of chemotherapy on the tumor immune microenvironment. We treat human liver metastases slices with 5-fluorouracil (5-FU) plus either irinotecan or oxaliplatin, then perform single-cell transcriptome analyses. Results from eight cases reveal two cellular subtypes with divergent responses to chemotherapy. Susceptible tumors are characterized by a stemness signature, an activated interferon pathway, and suppression of PD-1 ligands in response to 5-FU+irinotecan. Conversely, immune checkpoint TIM-3 ligands are maintained or upregulated by chemotherapy in CRC with an enterocyte-like signature, and combining chemotherapy with TIM-3 blockade leads to synergistic tumor killing. Our analyses highlight chemomodulation of the immune microenvironment and provide a framework for combined chemo-immunotherapies.