Multiparametric Analyses of Hepatocellular Carcinoma Somatic Mouse Models and Their Associated Tumor Microenvironment

Abstract

The rising incidence and increasing mortality of hepatocellular carcinoma (HCC), combined with its high tumor heterogeneity, lack of druggable targets, and tendency to develop resistance to chemotherapeutics, make the development of better models for this cancer an urgent challenge. To better mimic the high diversity within the HCC genetic landscape, versatile somatic murine models have recently been developed using the hydrodynamic tail vein injection (HDTVi) system. These represent novel in vivo tools to interrogate HCC phenotype and response to therapy, and importantly, allow further analyses of the associated tumor microenvironment (TME) shaped by distinct genetic backgrounds. Here, we describe several optimized protocols to generate, collect, and experimentally utilize various samples obtained from HCC somatic mouse models generated by HDTVi. More specifically, we focus on techniques relevant to ex vivo analyses of the complex liver TME using multiparameter flow cytometric analyses of over 21 markers, immunohistochemistry, immunofluorescence, and histochemistry. We describe the transcriptional assessment of whole tissue, or of isolated immune subsets by flow‐cytometry‐based cell sorting, and other protein‐oriented analyses. Together, these streamlined protocols allow the optimal use of each HCC murine model of interest and will assist researchers in deciphering the relations between cancer cell genetics and systemic and local changes in immune cell landscapes in the context of HCC progression. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Generation of HCC mouse models by hydrodynamic tail vein injection Basic Protocol 2: Assessment of HCC tumor progression by magnetic resonance imaging Basic Protocol 3: Mouse sacrifice and sample collection in HCC mouse models Support Protocol 1: Preparation of serum or plasma from blood Basic Protocol 4: Single‐cell preparation and HCC immune landscape phenotyping by flow cytometry Alternate Protocol 1: Flow cytometric analysis of circulating immune cells Support Protocol 2: Generation, maintenance, and characterization of HCC cell lines Support Protocol 3: Fluorescence‐activated cell sorting of liver single‐cell preparation Basic Protocol 5: Preparation and immunohistochemical analysis of tumor tissues from HCC‐bearing liver Alternate Protocol 2: Preparation and analyses for immunofluorescence staining of HCC‐bearing liver Support Protocol 4: Liver‐specific phenotypic analyses of liver sections Support Protocol 5: Immunohistochemical quantification in liver sections Basic Protocol 6: Preparation of snap‐frozen tumor tissue from extracted liver and transcriptional analyses of bulk tumor or sorted cells Alternate Protocol 3: Protein analyses from HCC samples and serum or plasma

Rational combination of SHP2 and mTOR inhibition for the treatment of hepatocellular carcinoma

“…In vivo, this aggressive HCC model quickly developed multinodular HCC tumors with dedifferentiated characteristics, as previously reported [25,58]. We then compared tumor growth in the presence of vehicle, AZD8055, RMC-4550 or the combination of the last two, in mice that developed HCC after hydrodynamic tail vein injection of the Myc OE ;Trp53 KO vectors, as described previously [26]. Strikingly, the combination of both drugs significantly improved the survival of the mice (Fig.…”

Ras/MAPK signalling intensity defines subclonal fitness in a mouse model of hepatocellular carcinoma

“…Mouse tumours were dissociated into single-cell suspension using the Mouse tumour dissociation kit (Miltenyi Biotec) and the gentleMACS Octo Dissociator following the manufacturer’s instructions and as described in Taranto et al, 2021 . The cell suspension was passed through a 70 μm cell strainer (Miltenyi), centrifuged at 300× g for 10 min at 4°C and washed three times in FACS buffer.…”

Cancer cell genetics shaping of the tumor microenvironment reveals myeloid cell-centric exploitable vulnerabilities in hepatocellular carcinoma

“…HCC somatic mouse models were generated using hydrodynamic tail vein injections (HDTVi) as previously described 47,105 . Briefly, a volume equivalent to 10% of mouse body weight of sterile 0.9% NaCl saline solution containing plasmid mixtures was injected into the mouse lateral tail vein in 7-10 seconds.…”

“…The pT/CaggsNras G12V -IRES-Luc vector 106 was kindly provided by Lars Zender (University of Tuebingen, Tuebingen, Germany), the pX330- Trp53 (Addgene 59910), the pX330-Pten (Addgene 59909), were previously validated and are publicly available. Mice were followed with weekly and bi-weekly MRI starting one-week post-HDTVi to measure the longitudinal progression of tumor volumes (as previously described 105,107 ). Animals were sacrificed when symptomatic and/or when tumor volume reached a total volume ≥2 cm 3 in survival curves and end-stage analyses (humane endpoint).…”

“…Blood was collected by heart puncture and subsequently transcardially perfused with PBS until liver is cleared of blood. Harvested livers were macro-dissected and used for further analysis 105 .…”