Mouse models of disease play a pivotal role at all stages of cancer drug development. Cell-line derived subcutaneous tumour models are predominant in early drug discovery, but there is growing recognition of the importance of the more complex orthotopic and metastatic tumour models for understanding both target biology in the correct tissue context, and the impact of the tumour microenvironment and the immune system in responses to treatment. The aim of this review is to highlight the value that orthotopic and metastatic models bring to the study of tumour biology and drug development while pointing out those models that are most likely to be encountered in the literature. Important developments in orthotopic models, such as the increasing use of early passage patient material (PDXs, organoids) and humanised mouse models are discussed, as these approaches have the potential to increase the predictive value of preclinical studies, and ultimately improve the success rate of anticancer drugs in clinical trials.

Supplementary Methods; Supplementary Figures S1-S5

Supplementary Tables S1-S2.

Carcinogen-induced lung cancer in A/J mice is an established model of tumor initiation and development. The impact of radiation, immune response, and STING pathway activation on tumor development in this model is not well understood. In this study, we examined the effect of radiation and the STING agonist, DMXAA (Vadimezan), on the development of urethane-induced lung cancer. A/J mice were treated with urethane (i.p.) to induce the development of lung tumors. Three days after urethane treatment, mice received 13 Gy thorax radiation treatment (RT)/mock RT, DMXAA (20 mg/kg i.p.)/vehicle, or a combination treatment (n = 5). Mice were euthanized after 6 hours to 7 days after treatment to assess early events in the lung with RNA-sequencing and flow cytometry, or 5 months after treatment to assess tumor growth and long-term changes to the immune microenvironment with multiplex immunofluorescence. Mice treated with urethane followed by RT had fewer lung surface tumors (p < 0.01) and reduced tumor area (p = 0.0732) compared to their mock RT controls. In parallel, mice that received urethane pre-treatment and RT had high numbers of tertiary lymphoid structures (TLSs; these contained T-cells, B-cells, MHCII+ APCs and CXCL13) compared to the mock RT control mice (p < 0.001). Analysis of immune cell contents of the TLS revealed a notable increase in CD4, CD8 and regulatory T-cells in the RT condition (with urethane pretreatment). Important early events in the lung following RT of urethane pre-treated mice included a transient increase in type I and type II interferons, CD4 and CD8 T-cell infiltration (p < 0.001) and a strong downregulation of cell cycle events. In contrast to these findings, DMXAA treatment after urethane resulted in fewer tumors (p < 0.05), but larger tumors compared to the vehicle-treated group. An increase in immune cells was observed in the lung, however, without clear structures that resembled TLSs. Early events following DMXAA treatment with urethane pre-treatment included dampened levels of type I IFN and TNF-α in circulation and a decrease in T-cell infiltration into the lungs. Interestingly, RT and DMXAA combination treatment (with urethane pre-treatment) drove a synergistic anti-tumor effect, further reducing tumor numbers compared to the RT or DMXAA controls 5 months after treatment. RT and STING agonist treatment of pre-neoplastic lesions have differential impacts on tumor growth in the A/J mouse model, and work synergistically when combined. The different outcomes of RT and DMXAA on tumor growth may be driven by their distinctive mechanism of action on immune responses and capacities to form TLSs. These findings may have future implications for strategies for the early treatment of lung and other cancers, and they suggest that immune responses, including modulation of the STING pathway, may be an important aspect of early tumor development that could be targeted therapeutically. Citation Format: Kay Shigemori, Yanyan Jiang, Bruno Beernaert, Anderson J. Ryan, Eileen E. Parkes. Radiation and STING activation limit tumor development and modulate the immune environment via distinct mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6406.

Background Radiotherapy treatment is a mainstay of cancer treatments including for thoracic malignancies such as lung or breast cancer. Cardiac toxicity is a recognised long-term complication of thoracic radiotherapy which persists despite improvements in therapeutic modalities. The mechanisms and potential therapeutic targets that could provide cardiac protection in the context of radiation therapy remain incompletely understood. Here we investigated early and late cardiac toxicity following irradiation using the A/J mouse model to identify potential molecular drivers. Methods Single doses of irradiation of either 13 or 15 Gy were delivered to female A/J mice aged 6 – 8 weeks using a SmART-PLAN system. ECG traces and analysis were performed on anaesthetised mice. Cardiac tissue was harvested up to 32 weeks following irradiation for histological analysis and second-harmonic imaging microscopy. Results Cardiac RT resulted in cardiac conductivity abnormalities including prolonged QTc interval. Additionally, an increase in pericardial and perivascular fibrosis was noted with a marked increase in pericardial fibrosis at 15 Gy compared to 13 Gy. Persistent DNA damage response was identified in cardiomyocytes at 7 days post irradiation and polarisation of cardiac-infiltrating macrophages towards a CD206+ M2-like phenotype at the same timepoint. Conclusions Our data introduce the A/J mouse as a novel model for the study of physiologically relevant early and late cardiac toxicities following irradiation. Early events that could contribute to long term toxicities include persistent DNA damage response and repolarisation of macrophages, further investigation of which could identify potential future cardioprotective therapeutic strategies.

Supplementary Figures S1-S2 from Vascular Endothelial Growth Factor Receptor-1 Contributes to Resistance to Anti–Epidermal Growth Factor Receptor Drugs in Human Cancer Cells

Supplemental Figures and Tables

ABSTRACT Dysregulation of the ERBB/EGFR signalling pathway causes multiple types of cancer. Accordingly, ADAM17, the primary shedding enzyme that releases and activates ERBB ligands, is tightly regulated. It has recently become clear that iRhom proteins, inactive members of the rhomboid-like superfamily, are regulatory cofactors for ADAM17. Here, we show that oncogenic KRAS mutants target the cytoplasmic domain of iRhom2 (also known as RHBDF2) to induce ADAM17-dependent shedding and the release of ERBB ligands. Activation of ERK1/2 by oncogenic KRAS induces the phosphorylation of iRhom2, recruitment of the phospho-binding 14-3-3 proteins, and consequent ADAM17-dependent shedding of ERBB ligands. In addition, cancer-associated mutations in iRhom2 act as sensitisers in this pathway by further increasing KRAS-induced shedding of ERBB ligands. This mechanism is conserved in lung cancer cells, where iRhom activity is required for tumour xenograft growth. In this context, the activity of oncogenic KRAS is modulated by the iRhom2-dependent release of ERBB ligands, thus placing the cytoplasmic domain of iRhom2 as a central component of a positive feedback loop in lung cancer cells. This article has an associated First Person interview with the first authors of the paper.

Tumor-bearing experimental animals are essential for preclinical cancer drug development. A broad range of tumor models is available, with the simplest and most widely used involving a tumor of mouse or human origin growing beneath the skin of a mouse: the subcutaneous tumor model. Here, we outline the different types of in vivo tumor model, including some of their advantages and disadvantages and how they fit into the drug-development process. We then describe in more detail the subcutaneous tumor model and key steps needed to establish it in the laboratory, namely: choosing the mouse strain and tumor cells; cell culture, preparation and injection of tumor cells; determining tumor volume; mouse welfare; and an appropriate experimental end point. The protocol leads to subcutaneous tumor growth usually within 1-3 weeks of cell injection and is suitable for those with experience in tissue culture and mouse experimentation.

This article has been retracted: please see Elsevier Policy on Article Withdrawal ( http://www.elsevier.com/locate/withdrawalpolicy ). This article has been retracted at the request of co-authors, Prof. Fairclough and Prof. Ryan, as the research data was incorrect and disagrees with the vast majority of the published literature. The corresponding author, Prof I.M. Ward is unfortunately now deceased and the other two co-authors, Prof. Lewis and Prof. Duckett are uncontactable due to retirement. Editors apologize to readers of the journal that this was not detected during the submission process.

Background The radiosensitising effect of the poly(ADP-ribose) polymerase inhibitor olaparib on tumours has been reported. However, its effect on normal tissues in combination with radiation has not been well studied. Herein, we investigated the therapeutic index of olaparib combined with hemithoracic radiation in a urethane-induced mouse lung cancer model. Methods To assess tolerability, A/J mice were treated with olaparib plus whole thorax radiation (13 Gy), body weight changes were monitored and normal tissue effects were assessed by histology. In anti-tumour (intervention) studies, A/J mice were injected with urethane to induce lung tumours, and were then treated with olaparib alone, left thorax radiation alone or the combination of olaparib plus left thorax radiation at 8 weeks (early intervention) or 18 weeks (late intervention) after urethane injection. Anti-tumour efficacy and normal tissue effects were assessed by visual inspection, magnetic resonance imaging and histology. Results Enhanced body weight loss and oesophageal toxicity were observed when olaparib was combined with whole thorax but not hemithorax radiation. In both the early and late intervention studies, olaparib increased the anti-tumour effects of hemithoracic irradiation without increasing lung toxicity. Conclusions The addition of olaparib increased the therapeutic index of hemithoracic radiation in a mouse model of lung cancer.

Abstract Insights into oncogenesis derived from cancer susceptibility loci (SNP) hold the potential to facilitate better cancer management and treatment through precision oncology. However, therapeutic insights have thus far been limited by our current lack of understanding regarding both interactions of these loci with somatic cancer driver mutations and their influence on tumorigenesis. For example, although both germline and somatic genetic variation to the p53 tumor suppressor pathway are known to promote tumorigenesis, little is known about the extent to which such variants cooperate to alter pathway activity. Here we hypothesize that cancer risk-associated germline variants interact with somatic TP53 mutational status to modify cancer risk, progression, and response to therapy. Focusing on a cancer risk SNP (rs78378222) with a well-documented ability to directly influence p53 activity as well as integration of germline datasets relating to cancer susceptibility with tumor data capturing somatically-acquired genetic variation provided supportive evidence for this hypothesis. Integration of germline and somatic genetic data enabled identification of a novel entry point for therapeutic manipulation of p53 activities. A cluster of cancer risk SNPs resulted in increased expression of prosurvival p53 target gene KITLG and attenuation of p53-mediated responses to genotoxic therapies, which were reversed by pharmacologic inhibition of the prosurvival c-KIT signal. Together, our results offer evidence of how cancer susceptibility SNPs can interact with cancer driver genes to affect cancer progression and identify novel combinatorial therapies. Significance: These results offer evidence of how cancer susceptibility SNPs can interact with cancer driver genes to affect cancer progression and present novel therapeutic targets.

We recently reported that genetic or pharmacological inhibition of insulin-like growth factor receptor (IGF-1R) slows DNA replication and induces replication stress by downregulating the regulatory subunit RRM2 of ribonucleotide reductase, perturbing deoxynucleotide triphosphate (dNTP) supply. Aiming to exploit this effect in therapy we performed a compound screen in five breast cancer cell lines with IGF neutralising antibody xentuzumab. Inhibitor of checkpoint kinase CHK1 was identified as a top screen hit. Co-inhibition of IGF and CHK1 caused synergistic suppression of cell viability, cell survival and tumour growth in 2D cell culture, 3D spheroid cultures and in vivo. Investigating the mechanism of synthetic lethality, we reveal that CHK1 inhibition in IGF-1R depleted or inhibited cells further downregulated RRM2, reduced dNTP supply and profoundly delayed replication fork progression. These effects resulted in significant accumulation of unreplicated single-stranded DNA and increased cell death, indicative of replication catastrophe. Similar phenotypes were induced by IGF:WEE1 co-inhibition, also via exacerbation of RRM2 downregulation. Exogenous RRM2 expression rescued hallmarks of replication stress induced by co-inhibiting IGF with CHK1 or WEE1, identifying RRM2 as a critical target of the functional IGF:CHK1 and IGF:WEE1 interactions. These data identify novel therapeutic vulnerabilities and may inform future trials of IGF inhibitory drugs.

Cancer gene therapies are usually designed either to express wild-type copies of tumor suppressor genes or to exploit tumor-associated phenotypic changes to endow selective cytotoxicity. However, these approaches become less relevant to cancers that contain many independent mutations, and the situation is made more complex by our increased understanding of clonal evolution of tumors, meaning that different metastases and even regions of the same tumor mass have distinct mutational and phenotypic profiles. In contrast, the relatively genetically stable tumor microenvironment (TME) therefore provides an appealing therapeutic target, particularly since it plays an essential role in promoting cancer growth, immune tolerance, and acquired resistance to many therapies. Recently, a variety of different TME-targeted gene therapy and armed oncolytic strategies have been explored, with particular success observed in strategies targeting the cancer stroma, reducing tumor vasculature, and repolarizing the immunosuppressive microenvironment. Herein, we review the progress of these TME-targeting approaches and try to highlight those showing the greatest promise.

Abstract Inhibition of IGF receptor (IGF1R) delays repair of radiation-induced DNA double-strand breaks (DSB), prompting us to investigate whether IGF1R influences endogenous DNA damage. Here we demonstrate that IGF1R inhibition generates endogenous DNA lesions protected by 53BP1 bodies, indicating under-replicated DNA. In cancer cells, inhibition or depletion of IGF1R delayed replication fork progression accompanied by activation of ATR–CHK1 signaling and the intra-S-phase checkpoint. This phenotype reflected unanticipated regulation of global replication by IGF1 mediated via AKT, MEK/ERK, and JUN to influence expression of ribonucleotide reductase (RNR) subunit RRM2. Consequently, inhibition or depletion of IGF1R downregulated RRM2, compromising RNR function and perturbing dNTP supply. The resulting delay in fork progression and hallmarks of replication stress were rescued by RRM2 overexpression, confirming RRM2 as the critical factor through which IGF1 regulates replication. Suspecting existence of a backup pathway protecting from toxic sequelae of replication stress, targeted compound screens in breast cancer cells identified synergy between IGF inhibition and ATM loss. Reciprocal screens of ATM-proficient/deficient fibroblasts identified an IGF1R inhibitor as the top hit. IGF inhibition selectively compromised growth of ATM-null cells and spheroids and caused regression of ATM-null xenografts. This synthetic-lethal effect reflected conversion of single-stranded lesions in IGF-inhibited cells into toxic DSBs upon ATM inhibition. Overall, these data implicate IGF1R in alleviating replication stress, and the reciprocal IGF:ATM codependence we identify provides an approach to exploit this effect in ATM-deficient cancers. Significance: This study identifies regulation of ribonucleotide reductase function and dNTP supply by IGFs and demonstrates that IGF axis blockade induces replication stress and reciprocal codependence on ATM.

Dysregulation of the ERBB/EGFR signalling pathway causes multiple types of cancer (1, 2). Accordingly, ADAM17, the primary shedding enzyme that releases and activates ERBB ligands, is tightly regulated. It has recently become clear that iRhoms, inactive members of the rhomboid-like superfamily, are regulatory cofactors for ADAM17 (3, 4). Here we show that oncogenic KRAS mutants target the cytoplasmic domain of iRhom2 to induce ADAM17-dependent shedding and the release of ERBB ligands. Activation of ERK1/2 by oncogenic KRAS induces the phosphorylation of iRhom2, recruitment of the phospho-binding 14-3-3 proteins, and consequent ADAM17-dependent shedding of ERBB ligands. In addition, cancer-associated mutations in iRhom2 act as sensitisers in this pathway by further increasing KRAS-induced shedding of ERBB ligands. This mechanism is conserved in lung cancer cells, where iRhom activity is required for tumour xenograft growth. In this context, the activity of oncogenic KRAS is modulated by the iRhom2-dependent release of ERBB ligands, thus placing iRhom2 as a central component of a positive feedback loop in lung cancer cells. Overall, the cytoplasmic domain of iRhom2 is a critical component of KRAS-induced oncogenesis of lung cancer cells. Both ADAM17 and iRhom2 have also been implicated in a wide range of other cancers (5–10), so the mechanism we have revealed may also have wider oncogenic significance.

Radiation-induced DNA double-strand breaks (DSBs) can be repaired by homologous recombination (HR) and nonhomologous end joining (NHEJ). Recently, it has been found that chronic tumor hypoxia compromises HR repair of DNA DSBs but activates the NHEJ protein DNAPK. We therefore hypothesized that inhibition of DNAPK can preferentially potentiate the sensitivity of chronically hypoxic cancer cells to radiation through contextual synthetic lethality in vivo. In this study, we investigated the impact of DNAPK inhibition by a novel selective DNAPK inhibitor, NU5455, on the repair of radiation-induced DNA DSBs in chronically hypoxic and nonhypoxic cells across a range of xenograft models. We found that NU5455 inhibited DSB repair following radiation in both chronically hypoxic and nonhypoxic tumor cells. Most importantly, the inhibitory effect was more pronounced in chronically hypoxic tumor cells than in nonhypoxic tumor cells. This is the first in vivo study to indicate that DNAPK inhibition may preferentially sensitize chronically hypoxic tumor cells to radiotherapy, suggesting a broader therapeutic window for transient DNAPK inhibition combined with radiotherapy.

Inflammation in macrophages and pancreatic islets is a hallmark of both type 1 diabetes (T1D) and type 2 diabetes (T2D). The enzyme 12-lipoxygenase (12-LOX) is expressed in macrophages and β cells, produces the eicosanoid 12(S)-HETE and promotes inflammation. GPR31 has recently been identified as the 12(S)-HETE receptor. To clarify the role of GPR31 in diabetic inflammation, we generated Gpr31b-/- mice on the C57BL/6J background. Gpr31-/- mice are viable, with normal body weight, glucose tolerance, and β-cell mass, similar to 12-LOX knockout mice. Upon low dose streptozotocin treatment to induce β-cell inflammation, Gpr31b-/- mice remained normoglycemic unlike wildtype littermates, and exhibited no reductions in β-cell mass. Isolated islets treated with the insulin receptor antagonist S961 to mimic insulin resistance showed the expected decrease in Ins1, Pdx1, and Gpx1 mRNA levels. By contrast, in Gpr31b-/- islets, these genes remained unaltered. Additionally, Gpr31b-/- islets showed a trend towards a decrease in ER stress markers Ddit3 and spliced-Xbp1 expression. To examine GPR31 in macrophage inflammation, we first interrogated its role in the generation of proinflammatory macrophages upon polarization in vitro to the M1 state; under these conditions, we found no differences between Gpr31b-/- and wildtype macrophages, suggesting that GPR31 does not play a role in macrophage polarization. To test a role for GPR31 in proinflammatory macrophage function, we leveraged a zebrafish tailfin injury model, in which macrophages migrate to the site of tailfin injury. Compared to control fish, fish harboring knockdown of GPR31 showed reduced numbers of macrophages at the site of injury. Taken together, our data support the notion that the effects of 12-LOX are mediated through GPR31, and that GPR31 appears to promote proinflammatory responses in both macrophages and pancreatic islets. Disclosure S. A. Tersey: None. M. Walsh: None. M. Hernandez-perez: None. J. Dowgielewicz: None. A. Kulkarni: None. R. Anderson: None. R. G. Mirmira: Advisory Panel; Self; Hibercell Inc., Sigilon Therapeutics, Inc., Veralox Therapeutics, Employee; Spouse/Partner; Beta Bionics, Inc. Funding National Center for Advancing Translational Sciences (R03TR003381)