Advances in Making Cancer Mouse Models More Accessible and Informative through Non-Germline Genetic Engineering.

Genetically engineered mouse models (GEMMs) allow for modeling of spontaneous tumorigenesis within its native microenvironment in mice and have provided invaluable insights into mechanisms of tumorigenesis and therapeutic strategies to treat human disease. However, as their generation requires germline manipulation and extensive animal breeding that is time-, labor-, and cost-intensive, traditional GEMMs are not accessible to most researchers, and fail to model the full breadth of cancer-associated genetic alterations and therapeutic targets. Recent advances in genome-editing technologies and their implementation in somatic tissues of mice have ushered in a new class of mouse models: non-germline GEMMs (nGEMMs). nGEMM approaches can be leveraged to generate somatic tumors de novo harboring virtually any individual or group of genetic alterations found in human cancer in a mouse through simple procedures that do not require breeding, greatly increasing the accessibility and speed and scale on which GEMMs can be produced. Here we describe the technologies and delivery systems used to create nGEMMs and highlight new biological insights derived from these models that have rapidly informed functional cancer genomics, precision medicine, and immune oncology.

Nanoparticle delivery of innate immune agonists combines with senescence-inducing agents to mediate T cell control of pancreatic cancer.

Pancreatic ductal adenocarcinoma has quickly risen to become the 3rd leading cause of cancer-related death. This is in part due to its fibrotic tumor microenvironment (TME) that contributes to poor vascularization and immune infiltration and subsequent chemo- and immunotherapy failure. Here we investigated an innovative immunotherapy approach combining local delivery of STING and TLR4 innate immune agonists via lipid-based nanoparticles (NPs) co-encapsulation with senescence-inducing RAS-targeted therapies that can remodel the immune suppressive PDAC TME through the senescence-associated secretory phenotype. Treatment of transplanted and autochthonous PDAC mouse models with these regimens led to enhanced uptake of NPs by multiple cell types in the PDAC TME, induction of type I interferon and other pro-inflammatory signaling, increased antigen presentation by tumor cells and antigen presenting cells, and subsequent activation of both innate and adaptive immune responses. This two-pronged approach produced potent T cell-driven and Type I interferon-dependent tumor regressions and long-term survival in preclinical PDAC models. STING and TLR4-mediated Type I interferon signaling were also associated with enhanced NK and CD8+ T cell immunity in human PDAC. Thus, combining localized immune agonist delivery with systemic tumor-targeted therapy can synergize to orchestrate a coordinated innate and adaptive immune assault to overcome immune suppression and activate durable anti-tumor T cell responses against PDAC.

EZH2 inhibition remodels the inflammatory senescence-associated secretory phenotype to potentiate pancreatic cancer immune surveillance.

Immunotherapies that produce durable responses in some malignancies have failed in pancreatic ductal adenocarcinoma (PDAC) due to rampant immune suppression and poor tumor immunogenicity. We and others have demonstrated that induction of the senescence-associated secretory phenotype (SASP) can be an effective approach to activate anti-tumor natural killer (NK) cell and T cell immunity. In the present study, we found that the pancreas tumor microenvironment suppresses NK cell and T cell surveillance after therapy-induced senescence through enhancer of zeste homolog 2 (EZH2)-mediated epigenetic repression of proinflammatory SASP genes. EZH2 blockade stimulated production of SASP chemokines CCL2 and CXCL9/10, leading to enhanced NK cell and T cell infiltration and PDAC eradication in mouse models. EZH2 activity was also associated with suppression of chemokine signaling and cytotoxic lymphocytes and reduced survival in patients with PDAC. These results demonstrate that EZH2 represses the proinflammatory SASP and that EZH2 inhibition combined with senescence-inducing therapy could be a powerful means to achieve immune-mediated tumor control in PDAC.

Breast cancer prevention by short-term inhibition of TGFß signaling.

Cancer prevention has a profound impact on cancer-associated mortality and morbidity. We previously identified TGFß signaling as a candidate regulator of mammary epithelial cells associated with breast cancer risk. Here, we show that short-term TGFBR inhibitor (TGFBRi) treatment of peripubertal ACI inbred and Sprague Dawley outbred rats induces lasting changes and prevents estrogen- and carcinogen-induced mammary tumors, respectively. We identify TGFBRi-responsive cell populations by single cell RNA-sequencing, including a unique epithelial subpopulation designated secretory basal cells (SBCs) with progenitor features. We detect SBCs in normal human breast tissues and find them to be associated with breast cancer risk. Interactome analysis identifies SBCs as the most interactive cell population and the main source of insulin-IGF signaling. Accordingly, inhibition of TGFBR and IGF1R decrease proliferation of organoid cultures. Our results reveal a critical role for TGFß in regulating mammary epithelial cells relevant to breast cancer and serve as a proof-of-principle cancer prevention strategy.

Insights into Immune Escape During Tumor Evolution and Response to Immunotherapy Using a Rat Model of Breast Cancer.

Animal models are critical for the preclinical validation of cancer immunotherapies. Unfortunately, mouse breast cancer models do not faithfully reproduce the molecular subtypes and immune environment of the human disease. In particular, there are no good murine models of estrogen receptor-positive (ER+) breast cancer, the predominant subtype in patients. Here, we show that Nitroso-N-methylurea-induced mammary tumors in outbred Sprague-Dawley rats recapitulate the heterogeneity for mutational profiles, ER expression, and immune evasive mechanisms observed in human breast cancer. We demonstrate the utility of this model for preclinical studies by dissecting mechanisms of response to immunotherapy using combination TGFBR inhibition and PD-L1 blockade. Short-term treatment of early-stage tumors induced durable responses. Gene expression profiling and spatial mapping classified tumors as inflammatory and noninflammatory, and identified IFNγ, T-cell receptor (TCR), and B-cell receptor (BCR) signaling, CD74/MHC II, and epithelium-interacting CD8+ T cells as markers of response, whereas the complement system, M2 macrophage phenotype, and translation in mitochondria were associated with resistance. We found that the expression of CD74 correlated with leukocyte fraction and TCR diversity in human breast cancer. We identified a subset of rat ER+ tumors marked by expression of antigen-processing genes that had an active immune environment and responded to treatment. A gene signature characteristic of these tumors predicted disease-free survival in patients with ER+ Luminal A breast cancer and overall survival in patients with metastatic breast cancer receiving anti-PD-L1 therapy. We demonstrate the usefulness of this preclinical model for immunotherapy and suggest examination to expand immunotherapy to a subset of patients with ER+ disease. See related Spotlight by Roussos Torres, p. 672.

The impact of tumor epithelial and microenvironmental heterogeneity on treatment responses in HER2+ breast cancer.

Despite the availability of multiple human epidermal growth factor receptor 2-targeted (HER2-targeted) treatments, therapeutic resistance in HER2+ breast cancer remains a clinical challenge. Intratumor heterogeneity for HER2 and resistance-conferring mutations in the PIK3CA gene (encoding PI3K catalytic subunit α) have been investigated in response and resistance to HER2-targeting agents, while the role of divergent cellular phenotypes and tumor epithelial-stromal cell interactions is less well understood. Here, we assessed the effect of intratumor cellular genetic heterogeneity for ERBB2 (encoding HER2) copy number and PIK3CA mutation on different types of neoadjuvant HER2-targeting therapies and clinical outcome in HER2+ breast cancer. We found that the frequency of cells lacking HER2 was a better predictor of response to HER2-targeted treatment than intratumor heterogeneity. We also compared the efficacy of different therapies in the same tumor using patient-derived xenograft models of heterogeneous HER2+ breast cancer and single-cell approaches. Stromal determinants were better predictors of response than tumor epithelial cells, and we identified alveolar epithelial and fibroblastic reticular cells as well as lymphatic vessel endothelial hyaluronan receptor 1-positive (Lyve1+) macrophages as putative drivers of therapeutic resistance. Our results demonstrate that both preexisting and acquired resistance to HER2-targeting agents involve multiple mechanisms including the tumor microenvironment. Furthermore, our data suggest that intratumor heterogeneity for HER2 should be incorporated into treatment design.

Subclonal cooperation drives metastasis by modulating local and systemic immune microenvironments.

Most human tumours are heterogeneous, composed of cellular clones with different properties present at variable frequencies. Highly heterogeneous tumours have poor clinical outcomes, yet the underlying mechanism remains poorly understood. Here, we show that minor subclones of breast cancer cells expressing IL11 and FIGF (VEGFD) cooperate to promote metastatic progression and generate polyclonal metastases composed of driver and neutral subclones. Expression profiling of the epithelial and stromal compartments of monoclonal and polyclonal primary and metastatic lesions revealed that this cooperation is indirect, mediated through the local and systemic microenvironments. We identified neutrophils as a leukocyte population stimulated by the IL11-expressing minor subclone and showed that the depletion of neutrophils prevents metastatic outgrowth. Single-cell RNA-seq of CD45+ cell populations from primary tumours, blood and lungs demonstrated that IL11 acts on bone-marrow-derived mesenchymal stromal cells, which induce pro-tumorigenic and pro-metastatic neutrophils. Our results indicate key roles for non-cell-autonomous drivers and minor subclones in metastasis.

Dietary Fatty Acid Modification for the Treatment of Aspirin-Exacerbated Respiratory Disease: A Prospective Pilot Trial.

BACKGROUND: The high levels of eicosanoid production and the clinical efficacy of leukotriene-modifying pharmacotherapies for patients with aspirin-exacerbated respiratory disease (AERD) suggest that other interventions targeting arachidonic acid dysregulation may also improve disease control. OBJECTIVE: To assess the utility of a high omega-3/low omega-6 diet for the treatment of AERD. METHODS: Prospective, nonblinded dietary intervention in 10 adult patients with AERD at Brigham and Women's Hospital in Boston, MA. The primary objective was for subjects to reduce dietary omega-6 fatty acid consumption to less than 4 g/d and increase omega-3 intake to more than 3 g/d. The primary outcome was change in urinary leukotriene E4, with changes in other eicosanoids, platelet activation, lung function, and patient-reported questionnaires also assessed. RESULTS: Of the 10 subjects who screened for the study, all 10 completed the dietary intervention. Urinary leukotriene E4 decreased by 0.17 ng/mg (95% CI, -0.29 to -0.04; P = .02) and tetranor prostaglandin D-M decreased by 0.66 ng/mg creatinine (95% CI, -1.21 to -0.11; P = .02). There was a 15.1-point reduction in the 22-item Sino-Nasal Outcome Test score (95% CI, -24.3 to -6.0; P = .01), a 0.27-point reduction in the 7-item Asthma Control Questionnaire score (95% CI, -0.52 to -0.03; P = .03), and no change in FEV1 % predicted (P = .92) or forced vital capacity % predicted (P = .74). All patients lost some weight over the 2-week intervention period, and there were no diet-associated adverse events. CONCLUSIONS: A high omega-3/low omega-6 diet may be an appropriate adjunct treatment option for patients with AERD.

Thymic stromal lymphopoietin controls prostaglandin D2 generation in patients with aspirin-exacerbated respiratory disease.

BACKGROUND: Prostaglandin (PG) D2 is the dominant COX product of mast cells and is an effector of aspirin-induced respiratory reactions in patients with aspirin-exacerbated respiratory disease (AERD). OBJECTIVE: We evaluated the role of the innate cytokine thymic stromal lymphopoietin (TSLP) acting on mast cells to generate PGD2 and facilitate tissue eosinophilia and nasal polyposis in patients with AERD. METHODS: Urinary eicosanoid levels were measured in aspirin-tolerant control subjects and patients with AERD. Nasal polyp specimens from patients with AERD and chronic rhinosinusitis were analyzed by using quantitative PCR, Western blotting, and immunohistochemistry. Human cord blood-and peripheral blood-derived mast cells were stimulated with TSLP in vitro to assess PGD2 generation. RESULTS: Urinary levels of a stable PGD2 metabolite (uPGD-M) were 2-fold higher in patients with AERD relative to those in control subjects and increased further during aspirin-induced reactions. Peak uPGD-M levels during aspirin reactions correlated with reductions in blood eosinophil counts and lung function and increases in nasal congestion. Mast cells sorted from nasal polyps expressed PGD2 synthase (hematopoietic PGD2 synthase) mRNA at higher levels than did eosinophils from the same tissue. Whole nasal polyp TSLP mRNA expression correlated strongly with mRNA encoding hematopoietic PGD2 synthase (r = .75), the mast cell-specific marker carboxypeptidase A3 (r = .74), and uPGD-M (r = 0.74). Levels of the cleaved active form of TSLP were increased in nasal polyps from patients with AERD relative to those in aspirin-tolerant control subjects. Recombinant TSLP induced PGD2 generation by cultured human mast cells. CONCLUSIONS: Our study demonstrates that mast cell-derived PGD2 is a major effector of type 2 immune responses driven by TSLP and suggests that dysregulation of this innate system contributes significantly to the pathophysiology of AERD.