Autochthonous Tumors Research Articles

Abstract B65: Defining the tumor immune landscape in a mouse model of high-grade serous carcinoma

Abstract Clinical efficacy of immune checkpoint therapy in high-grade serous carcinoma (HGSC) has been disappointing to date and highlights the need to better understand the relationship between HGSC and its associated immune microenvironment. Tractable model systems for interrogating the tumor microenvironment in HGSC could be used to address this unmet need. We have generated genetically engineered mouse models (GEMMs) of HGSC utilizing oviduct-restricted tamoxifen-regulated Cre recombinase to drive conditional inactivation of key tumor suppressor genes (Brca1, Trp53, Rb1, Nf1) observed to be recurrently altered in human HGSC samples. These autochthonous tumors arise from the murine fallopian tube (oviduct) in the context of an intact immune system. We employed RNA sequencing (RNA-seq) of bulk tumors from 19 mouse HGSCs to compare their gene expression profiles to those of normal oviduct and ovary tissues (4 each), and 6 mouse endometrioid-like oviductal carcinomas based on conditional inactivation of Apc and Pten. We employed immunogenomic analysis of the RNA-seq data to characterize the intratumoral infiltrate of 28 immune cell types in these samples in silico. The analysis revealed a prevalence of gene expression signatures associated with immunosuppressive cell types, including myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs) in the mouse HGSCs, not observed in the other sample groups. Human HGSCs with available RNA-seq data (from TCGA samples) showed similar enrichment of MDSC and Treg gene signatures in the subset of human HGSCs classified as mesenchymal or immunoreactive expression subtypes. To further validate the findings in the mouse HGSCs, we used immunohistochemistry to detect subsets of tumor-infiltrating lymphocytes (TILs) in the tumor tissues. Specifically, CD8+ TIL infiltrates recapitulated epithelial (E-TIL), stromal (S-TIL) and combined epithelial/stromal (ES-TIL) patterns described in human HGSCs. Moreover, FoxP3+ Tregs were frequently observed to be co-infiltrated with CD8+ TILs as expected in an immunosuppressive tumor microenvironment. Gene set enrichment analysis showed that NF-kappa B signaling is among the top five pathways upregulated in HGSCs versus normal oviducts, suggesting a plausible mechanism by which tumor cells can alter and respond to the microenvironment during tumor development. These studies provide an experimental paradigm for improving our understanding of the role of the tumor microenvironment in HGSC pathogenesis and may aid in efforts to enhance the efficacy of immunotherapies in this lethal disease. Citation Format: Kevin W. McCool, Rork Kuick, Yali Zhai, Zach Freeman, Rong Wu, Eric Fearon, Kathleen R. Cho. Defining the tumor immune landscape in a mouse model of high-grade serous carcinoma [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr B65.

Cancer Burden Is Controlled by Mural Cell-β3-Integrin Regulated Crosstalk with Tumor Cells

Enhanced blood vessel (BV) formation is thought to drive tumor growth through elevated nutrient delivery. However, this observation has overlooked potential roles for mural cells in directly affecting tumor growth independent of BV function. Here we provide clinical data correlating high percentages of mural-β3-integrin-negative tumor BVs with increased tumor sizes but no effect on BV numbers. Mural-β3-integrin loss also enhances tumor growth in implanted and autochthonous mouse tumor models with no detectable effects on BV numbers or function. At a molecular level, mural-cell β3-integrin loss enhances signaling via FAK-p-HGFR-p-Akt-p-p65, driving CXCL1, CCL2, and TIMP-1 production. In particular, mural-cell-derived CCL2 stimulates tumor cell MEK1-ERK1/2-ROCK2-dependent signaling and enhances tumor cell survival and tumor growth. Overall, our data indicate that mural cells can control tumor growth via paracrine signals regulated by β3-integrin, providing a previously unrecognized mechanism of cancer growth control.

A novel cancer immunotherapy utilizing autologous tumour tissue.

With the recent interest in personalized medicine for cancer patients and immune therapy, the field of cancer vaccines has been resurrected. Previous autologous, whole cell tumour vaccine trials have not produced convincing results due, in part to poor patient selection and inactivation methos that are harsh on the cells. These methods can alter protein structure and antigenic profiles making vaccine candidates ineffective in stimulating immune response to autochthonous tumour cells. We investigated a novel method for inactivating tumour cells that uses UVA/UVB light and riboflavin (vitamin B2) (RF+UV). RF+UV inactivates the tumour cells' ability to replicate, yet preserves tumour cell integrity and antigenicity. Our results demonstrate that proteins are preserved on the surface of RF+UV-inactivated tumour cells and that they are immunogenic via induction of dendritic cell maturation, increase in IFNγ production and generation of tumour cell-specific IgG. Moreover, when formulated with an adjuvant ('Innocell vaccine') and tested in different murine tumour primary and metastatic disease models, decreased tumour growth, decreased metastatic disease and prolonged survival were observed. In addition, immune cells obtained from tumour tissue following vaccination had decreased exhausted and regulatory T cells, suggesting that activation of intra-tumoural T cells may be playing a role leading to reduced tumour growth. These data suggest that the RF+UV inactivation of tumour cells may provide an efficacious method for generating autologous whole tumour cell vaccines for use in cancer patients.

Immunization against ROS1 by DNA Electroporation Impairs K-Ras-Driven Lung Adenocarcinomas ​.

Non-small cell lung cancer (NSCLC) is still the leading cause of cancer death worldwide. Despite the introduction of tyrosine kinase inhibitors and immunotherapeutic approaches, there is still an urgent need for novel strategies to improve patient survival. ROS1, a tyrosine kinase receptor endowed with oncoantigen features, is activated by chromosomal rearrangement or overexpression in NSCLC and in several tumor histotypes. In this work, we have exploited transgenic mice harboring the activated K-Ras oncogene (K-RasG12D) that spontaneously develop metastatic NSCLC as a preclinical model to test the efficacy of ROS1 immune targeting. Indeed, qPCR and immunohistochemical analyses revealed ROS1 overexpression in the autochthonous primary tumors and extrathoracic metastases developed by K-RasG12D mice and in a derived transplantable cell line. As proof of concept, we have evaluated the effects of the intramuscular electroporation (electrovaccination) of plasmids coding for mouse- and human-ROS1 on the progression of these NSCLC models. A significant increase in survival was observed in ROS1-electrovaccinated mice challenged with the transplantable cell line. It is worth noting that tumors were completely rejected, and immune memory was achieved, albeit only in a few mice. Most importantly, ROS1 electrovaccination was also found to be effective in slowing the development of autochthonous NSCLC in K-RasG12D mice.

VARIETY OF TUMOR MODELS FOR TESTING ANTITUM TREATMENT ACTIVITY OF SUBSTANCES IN MICE

Selection of tolerated and toxic doses, schemes of therapy for new anticancer drugs, assessment of its toxicity, pharmacokinetics, metabolism, study of the mechanism of action and tumor sensitivity are carried out using models of human tumors in mice. The review contains a diverse description of experimental models (transplantable, genetically engineered, humanized, autochthonous, orthotopic, heterotopic and metastatic) tumors using laboratory mice. The advantages, disadvantages, directions and specific features of the use of mouse models, their role in the study of mechanisms of action of antitumor drugs are considered on the examples of recent research.

Antibody blockade of semaphorin 4D to sensitize pancreatic cancer to immune checkpoint blockade.

26 Background: Pancreatic ductal adenocarcinoma (PDAC) carries a dismal prognosis, and will soon become the second leading cause of cancer mortality. Unfortunately, T-cell directed immunotherapies have failed to demonstrate efficacy in PDAC. These failures may in part be mediated by an immunosuppressive tumor microenvironment (TME). Semaphorin 4D (Sema4D) is a glycoprotein which binds its cognate receptors Plexin B1/B2. Here we present our work in blocking Sema4D in a murine model of PDAC. Methods: C57b/6 mice were orthotopically injected with PDAC line (KP2) derived from KRASG12D,TP53Flox/Wt;P48-Cre autochthonous tumors and confirmed for disease by ultrasound. Mice were treated with FOLFIRINOX (5-FU, Irinotecan, Oxaliplatin, weekly), immune checkpoint blockade (ICB) (anti-PD1, anti-CTLA-4 mAbs bi-weekly), and anti-Sema4D mAB (bi-weekly). Human and mouse circulating and tumor infiltrating leukocytes were interrogated through flow cytometry (FACS) for immune subset and expression of Sema4D and Plexin receptors. Archived human PDAC tissues were assessed through quantitative immunohistochemistry (IHC) for presence of Sema4D positive infiltrate. Results: Both FACS and IHC analysis of human PDAC specimens confirm the presence and increased prevalence over normal pancreata of Sema4D lymphocytes and Plexin B1/B2 expressing tumor associated macrophages (TAMs). KP2 orthotopically injected mice exhibited longer survival when treated with the triple combination of FOLFIRINOX, ICB, and anti-Sema4D antibody, compared to FOLFIRINOX alone, FOLFIRINOX plus ICB, or FOLFIRINOX plus anti-Sema4D antibody (P < 0.02). Flow cytometric analysis of anti-Sema4D and ICB treated murine tumors show a doubling of penetration by CD 8+ effector T cells within tumors compared to control groups (P = 0.03). A loss in Sema4D fluorescence signal via FACS in tumor-infiltrating CD3+ leukocytes was observed in mice treated with anti-SEMA4D, confirming penetration and target blockade within the TME. Conclusions: Sema4D and Plexin B1/B2 leukocytes penetrate human PDAC tumors, and treatment with Sema4D blocking antibody improved response to ICB in combination with standard of care FOLFIRINOX in preclinical murine studies.

Abstract B48: Targeting the semaphorin 4D-plexin B axis to augment FOLFIRINOX in a murine model of pancreatic adenocarcinoma

Abstract Background: Pancreatic adenocarcinoma (PDAC) is the fourth leading cause of cancer-related mortality, with dismal 5-year prognosis of 8%, in part due to poor response to available therapies. Thus, new systemic cancer control therapies are in dire need. Semaphorin 4D (SEMA4D) is a soluble and membrane bound glycoprotein that binds its cognate Plexin B1/B2 receptors, expressed on monocytic and granulocytic leukocytes. Prior work has shown that increased expression of SEMA4D and Plexin B in resected PDAC patients is associated with lymph node and distant metastasis, as well as a worse prognosis. Additionally, SEMA4D blockade has conferred improved immune checkpoint blockade response in murine models of colorectal carcinoma and head and neck squamous cell carcinoma via abrogation of myeloid-derived suppressor cell recruitment and function, as well as enhanced T-cell recruitment and activity within the tumor microenvironment. Here we study the effects of SEMA4D blockade in a murine model of PDAC. Methods: C57b/6 mice were orthotopically injected with murine PDAC line (KP2) derived from KRASG12D,TP53Flox/Wt;P48-Cre autochthonous tumors. Mice were treated with Folfirinox (5-FU, irinotecan, oxaliplatin, weekly), immune checkpoint blockade (ICB) (anti-PD1, anti-CTLA-4 mAbs bi-weekly), and anti-SEMA4D mAB (biweekly). Peripheral blood and tumor-infiltrating leukocytes from patients with PDAC undergoing pancreaticoduodenectomy were assessed for Plexin B1 via flow cytometry. Results: Human PDAC demonstrates penetration of Plexin B1 positive leukocytes, most notably tumor-associated macrophages, neutrophils, and monocytes. Mice injected with KP2 developed tumors detectable via high-frequency ultrasound and exhibited longer survival when treated with the combination of Folfirinox, ICB, and anti-SEMA4D antibody, compared to Folfirinox alone, Folfirinox plus ICB, or Folfirinox plus anti-SEMA4D antibody. Conclusions: Plexin B1+ myeloid subsets penetrate human PDAC tumors, and treatment with SEMA4D-blocking antibody improved response to ICB in combination with standard-of-care Folfirinox in preclinical murine studies. Future work will focus on understanding the immune mechanism of improved therapeutic response, as well as further characterization of the prevalence and prognostic ramifications of the SEMA4D-Plexin B axis in PDAC. Citation Format: Luis I. Ruffolo, Katherine M. Jackson, Nicholas Ullman, Alexander Chacon, Alexa Melucci, Paul Burchard, Mary Georger, Rachel Jewell, Peter Prieto, Brian Belt, Crystal Mallow, Elizabeth Evans, Terrence Fisher, Maurice Zauderer, Christina Wu, Brian Olson, Gregory B. Lesinski, David C. Linehan. Targeting the semaphorin 4D-plexin B axis to augment FOLFIRINOX in a murine model of pancreatic adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr B48.

Faithful preclinical mouse models for better translation to bedside in the field of immuno-oncology.

The success of immunotherapy using immune checkpoint inhibitors has changed the practice of cancer treatment tremendously. However, there are still many clinical challenges, such as drug resistance, predictive biomarker development, exploration of combination therapies, and prediction of immune-related adverse events in preclinical settings. To overcome these problems, it is essential to establish faithful preclinical mouse models that recapitulate the clinical features, molecular genetics, biological heterogeneity, and immune microenvironment of human cancers. Here we review the advantages and disadvantages of current preclinical mouse models, including syngeneic murine tumor cell lines, autochthonous tumor models, cancer cell line-derived xenografts, patient-derived-xenografts, and various kinds of immunologically humanized mice. We discuss how these models should be characterized and applied in preclinical settings, and how we should prepare preclinical studies for successful translation from bench to bedside.

Abstract 4629: FOXA2 controls tumor-associated inflammation inKRAS-mutant lung cancer

Abstract Background: Although the role of the transcription factor FOXA2 in lung cancer has been investigated using mouse and human lung cancer cell lines, to our knowledge, the role of FOXA2 in autochthonous lung tumors in GEMM (Genetically Engineered Mouse Model) has not been determined. Methods: In order to determine the role of FOXA2 in autochthonous lung tumor cells, we deleted FOXA2 in lung epithelia of Kras-mutant lung cancer model mice (Scgb1a1-Cre;[tetO]-Kras4bG12D;Foxa2flox/flox, a GEMM) and assessed survival and lung histology of the GEMM. Results: Deletion of FOXA2 in autochthonous Kras-mutant lung tumor cells significantly extended the survival of Kras-mutant lung cancer model mice (median survival from 8 weeks to 23 weeks; n>14 for each group). Histological analysis indicated that FOXA2 was required for the growth of lung tumor cells and the recruitment of tumor-associated macrophages. Conclusion: FOXA2 promotes primary KRAS-mutant lung tumors in part by controlling tumor-associated inflammation. Citation Format: Koichi Tomoshige, Minzhe Guo, Iris Fink-Baldauf, William Stuart, Yutaka Maeda. FOXA2 controls tumor-associated inflammation inKRAS-mutant lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4629.

Abstract 2363: Targeting squamous lung tumor heterogeneity with EZH2 inhibition to improve immunotherapy responses

Abstract Immunotherapies, including those targeting the PD1/PD-L1 immune checkpoint, have become popular treatment options, but they are effective for only 20% of squamous lung cancer patients. Work in our laboratory has suggested that inhibition of the histone methyltransferase EZH2 drives a basal-like transcriptional state which includes expression of the immunotherapy target PD-L1, and work from others has demonstrated that EZH2 inhibition could influence the tumor microenvironment. Therefore, we hypothesize that combining EZH2 inhibition with anti-PD1 therapy could boost response in squamous lung cancers by targeting both the tumor and the microenvironmental heterogeneity. Our laboratory uses genetically engineered mouse models of lung cancer and patient-derived cell lines and organoids to dissect the mechanistic effects of EZH2 inhibition on tumor cells and immune cells. First, we treated a panel of squamous and adeno-squamous human NSCLC cell lines with EZH2 inhibition and observed increased expression of PD-L1 and another basal cell marker, NGFR, by flow cytometry. Similar results were obtained by treating two patient-derived organoids of squamous lung cancer with EZH2 inhibitor, suggesting that EZH2 inhibition drives squamous tumor cells into a more basal-like state which may be more susceptible to anti-PD1 immunotherapy. In our mouse models of squamous lung cancer, we noted abundant tumor-associated neutrophils, which we believe may be inhibiting efficacy of immunotherapy by suppressing CD8 T cell function. In support of the idea that inhibiting EZH2 will ameliorate this phenotype, lung neutrophils isolated from EZH2 conditional knock-out mice had significantly lower levels of the T cell suppressive protein Arginase 1 than WT lung neutrophils or Lkb1/Pten TANs. Lastly, we treated mice harboring autochthonous squamous lung tumors with EZH2 inhibitor, and observed a striking increase in the proportion of tumor cells expressing PD-L1 but now defect in tumor growth. In contrast, two squamous lung tumor bearing mice treated with EZH2 inhibitor and anti-PD-1 immunotherapy had tumor regression that was durable up to 4 weeks post-treatment initiation. We plan to expand the cohorts of mice and test this promising combination therapy in a variety of lung cancer mouse models. Completion of these studies will solidify the efficacy of a promising therapeutic combination and uncover mechanisms by which tumor hierarchies and microenvironments are changed by EZH2 inhibitors in squamous lung cancers. Given that one arm of a Phase 1/2 clinical trial combining EZH2 inhibition with anti-PDL1 just began recruiting late stage non-small cell lung cancer patients, learning the phenotypes and mechanisms of responders and non-responders will be extremely timely for any Phase 2/3 trials that ensue. Funded by V Foundation Scholar Award, K22 CA201036 and KY LCRP to CFB Citation Format: Tanner J. DuCote, Christine Fillmore Brainson. Targeting squamous lung tumor heterogeneity with EZH2 inhibition to improve immunotherapy responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2363.

Selective EGLN Inhibition Enables Ablative Radiotherapy and Improves Survival in Unresectable Pancreatic Cancer.

When pancreatic cancer cannot be removed surgically, patients frequently experience morbidity and death from progression of their primary tumor. Radiation therapy (RT) cannot yet substitute for an operation because radiation causes fatal bleeding and ulceration of the nearby stomach and intestines before achieving tumor control. There are no FDA-approved medications that prevent or reduce radiation-induced gastrointestinal injury. Here, we overcome this fundamental problem of anatomy and biology with the use of the oral EGLN inhibitor FG-4592, which selectively protects the intestinal tract from radiation toxicity without protecting tumors. A total of 70 KPC mice with autochthonous pancreatic tumors received oral FG-4592 or vehicle control ± ablative RT to a cumulative 75 Gy administered in 15 daily fractions to a limited tumor field. Although ablative RT reduced complications from local tumor progression, fatal gastrointestinal bleeding was observed in 56% of mice that received high-dose RT with vehicle control. However, radiation-induced bleeding was completely ameliorated in mice that received high-dose RT with FG-4592 (0% bleeding, P < 0.0001 compared with vehicle). Furthermore, FG-4592 reduced epithelial apoptosis by half (P = 0.002) and increased intestinal microvessel density by 80% compared with vehicle controls. EGLN inhibition did not stimulate cancer growth, as treatment with FG-4592 alone, or overexpression of HIF2 within KPC tumors independently improved survival. Thus, we provide a proof of concept for the selective protection of the intestinal tract by the EGLN inhibition to enable ablative doses of cytotoxic therapy in unresectable pancreatic cancer by reducing untoward morbidity and death from radiation-induced gastrointestinal bleeding. SIGNIFICANCE: Selective protection of the intestinal tract by EGLN inhibition enables potentially definitive doses of radiation therapy. This might allow radiation to be a surgical surrogate for unresectable pancreatic cancer.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/9/2327/F1.large.jpg.

Non-small cell lung cancer tumor microenvironment induces Arginase1 in neutrophils via the Toll-like receptor 2 signaling pathway

Abstract Neutrophils dominate the tumor microenvironment of non-small cell lung cancer. Previous studies show neutrophils suppress T cells and promote immunosuppression. The objective of the current study is to identify how tumor associated neutrophils (TAN) adopt the T-cell suppressive entity after being recruited into the tumor microenvironment. In the murine lung squamous cell carcinoma autochthonous tumor model (AdCre-Lkb1fl/fl, Ptenfl/fl; LP) and subcutaneous tumor model (Lewis lung carcinoma cell line; LLC), we found that Arginase 1 (Arg1) mRNA level in TAN is drastically higher than neutrophils from the bone marrow, spleen and peripheral blood. Arg1 suppresses T cell through Arginine depletion. Whole tumor lysates from LP tumor and LLC tumor induce Arg1 level in bone marrow-derived and peripheral neutrophils from healthy B6 mice in vitro, suggesting that the tumor microenvironment alters neutrophil properties. To identify the putative molecule(s) that induce Arg1 in TAN, we used fast protein liquid chromatography (FPLC) and mass spectrometry to analyze the whole tumor lysates from LP tumor and LLC tumor. Annexin A1 and 2 are among the common molecules that appear in Arg1-inducing FPLC fraction in both LP and LLC tumor lysates. Annexin A1, A2 are involved in toll-like receptor signaling. Flow cytometry showed more than 70% of Arg1+ TAN are either TLR2 or TLR4 positive. Interestingly, only TLR2 but not TLR4 inhibition blocked Arg1 induction by whole tumor lysates in naïve neutrophils. Taken together, tumor microenvironment induces Arg1 level in neutrophils in a TLR2 dependent manner. TLR2 is a putative therapeutic target for blocking tumor associated neutrophil-mediated immunosuppression in non-small cell lung cancer.

EGFR-targeted therapy alters the tumor microenvironment in EGFR-driven lung tumors: Implications for combination therapies.

Immune checkpoint inhibitors targeting the programmed cell death receptor/ligand 1 (PD-1/PD-L1) pathway have profoundly improved the clinical management of non-small-cell lung cancer (NSCLC). Nevertheless, the superiority of single-agent PD-1/PD-L1 inhibitors in pretreated EGFR mutant patients has turned out to be moderate. One proposed mechanism for poor response to immune checkpoint inhibitors is an immunosuppressive tumor microenvironment. Therefore, we utilized two autochthonous EGFR-driven lung tumor models to investigate dynamic microenvironmental responses to EGFR-TKI treatment. We observed that at an early stage, sensitive EGFR-TKIs caused obvious tumor shrinkage accompanied by increased cytotoxic CD8+ T cells and dendritic cells, eradication of Foxp3+ Tregs, and inhibition of M2-like polarization of macrophages. However, the tumor microenvironmental changes that may be most beneficial for combination treatment with immune-mediated anticancer approaches were only temporary and disappeared as treatment continued. Meanwhile, the level of myeloid-derived suppressor cells (MDSCs), particularly mononuclear MDSCs, was consistently elevated throughout the treatment. Analysis of inflammatory factors in serum showed that EGFR-TKIs increased the levels of IL-10 and CCL-2. Our study systematically analyzed dynamic changes in tumor microenvironments responding to EGFR-TKIs in vivo. The results have implications for combination therapy using EGFR-TKIs. The optimal sequence of the treatment and strategies that modulate the tumor microenvironment to a state that may favor antitumor immune responses need to be considered when designing clinical trials.

The Combination of MEK Inhibitor With Immunomodulatory Antibodies Targeting Programmed Death 1 and Programmed Death Ligand 1 Results in Prolonged Survival in Kras/p53-Driven Lung Cancer

IntroductionThis study aimed to characterize the tumor-infiltrating immune cells population in Kras/tumor protein 53 (Trp53)-driven lung tumors and to evaluate the combinatorial antitumor effect with MEK inhibitor (MEKi), trametinib, and immunomodulatory monoclonal antibodies (mAbs) targeting either programmed death -1 (PD-1) or programmed cell death ligand 1 (PD-L1) in vivo. MethodsTrp53FloxFlox;KrasG12D/+;Rosa26LSL-Luciferase/LSL-Luciferase (PKL) genetically engineered mice were used to develop autochthonous lung tumors with intratracheal delivery of adenoviral Cre recombinase. Using these tumor-bearing lungs, tumor-infiltrating immune cells were characterized by both mass cytometry and flow cytometry. PKL-mediated immunocompetent syngeneic and transgenic lung cancer mouse models were treated with MEKi alone as well as in combination with either anti–PD-1 or anti–PD-L1 mAbs. Tumor growth and survival outcome were assessed. Finally, immune cell populations within spleens and tumors were evaluated by flow cytometry and immunohistochemistry. ResultsMyeloid-derived suppressor cells (MDSCs) were significantly augmented in PKL-driven lung tumors compared to normal lungs of tumor-free mice. PD-L1 expression appeared to be highly positive in both lung tumor cells and, particularly MDSCs. The combinatory administration of MEKi with either anti–PD-1 or anti–PD-L1 mAbs synergistically increased antitumor response and survival outcome compared with single-agent therapy in both the PKL-mediated syngeneic and transgenic lung cancer models. Theses combinational treatments resulted in significant increases of tumor-infiltrating CD8+ and CD4+ T cells, whereas attenuation of CD11b+/Gr-1high MDSCs, in particular, Ly6Ghigh polymorphonuclear-MDSCs in the syngeneic model. ConclusionsThese findings suggest a potential therapeutic approach for untargetable Kras/p53-driven lung cancers with synergy between targeted therapy using MEKi and immunotherapies.

Cardio-Respiratory synchronized bSSFP MRI for high throughput in vivo lung tumour quantification

The identification and measurement of tumours is a key requirement in the study of tumour development in mouse models of human cancer. Disease burden in autochthonous tumours, such as those arising in the lung, can be seen with non-invasive imaging, but cannot be accurately measured using standard tools such as callipers. Lung imaging is further complicated in the mouse due to instabilities arising from the rapid but cyclic cardio-respiratory motions, and the desire to use free-breathing animals. Female A/JOlaHsd mice were either injected (i.p.) with PBS 0.1ml/10g body weight (n = 6), or 10% urethane/PBS 0.1ml/10g body weight (n = 12) to induce autochthonous lung tumours. Cardio-respiratory synchronised bSSFP MRI, at 200 μm isotropic resolution was performed at 8, 13 and 18 weeks post induction. Images from the same mouse at different time points were aligned using threshold-based segmented masks of the lungs (ITK-SNAP and MATLAB) and tumour volumes were determined via threshold-based segmentation (ITK-SNAP).Scan times were routinely below 10 minutes and tumours were readily identifiable. Image registration allowed serial measurement of tumour volumes as small as 0.056 mm3. Repetitive imaging did not lead to mouse welfare issues. We have developed a motion desensitised scan that enables high sensitivity MRI to be performed with high throughput capability of greater than 4 mice/hour. Image segmentation and registration allows serial measurement of individual, small tumours. This allows fast and highly efficient volumetric lung tumour monitoring in cohorts of 30 mice per imaging time point. As a result, adaptive trial study designs can be achieved, optimizing experimental and welfare outcomes.

IRGD-guided Tumor-penetrating Nanocomplexes for Therapeutic siRNA Delivery to Pancreatic Cancer.

Pancreatic cancer is one of the leading causes of cancer-related death, with 5-year survival of 8.5%. The lack of significant progress in improving therapy reflects our inability to overcome the desmoplastic stromal barrier in pancreatic ductal adenocarcinoma (PDAC) as well as a paucity of new approaches targeting its genetic underpinnings. RNA interference holds promise in targeting key mutations driving PDAC; however, a nucleic acid delivery vehicle that homes to PDAC and breaches the stroma does not yet exist. Noting that the cyclic peptide iRGD mediates tumor targeting and penetration through interactions with αvβ3/5 integrins and neuropilin-1, we hypothesized that "tandem" peptides combining a cell-penetrating peptide and iRGD can encapsulate siRNA to form tumor-penetrating nanocomplexes (TPN) capable of delivering siRNA to PDAC. The use of directly conjugated iRGD is justified by receptor expression patterns in human PDAC biopsies. In this work, we optimize iRGD TPNs with polyethylene glycol (PEG)-peptide conjugates for systemic delivery to sites of disease. We show that TPNs effectively knockdown siRNA targets in PDAC cell lines and in an immunocompetent genetically engineered mouse model of PDAC. Furthermore, we validate their tumor-penetrating ability in three-dimensional organoids and autochthonous tumors. In murine therapeutic trials, TPNs delivering anti-Kras siRNA significantly delay tumor growth. Thus, iRGD TPNs hold promise in treating PDAC by not only overcoming physical barriers to therapy, but by leveraging the stroma to achieve knockdown of the gold-standard genetic target. Moreover, the modular construction of this delivery platform allows for facile adaptation to future genetic target candidates in pancreatic cancer. Mol Cancer Ther; 17(11); 2377-88. ©2018 AACR.

Highlights of This Issue

NF2 is an important tumor suppressor and regulator of physical stresses and loss of NF2 cause NF2-related cancers (NF2 syndrome and mesothelioma). However, there is no proper treatment for these diseases until now. This study by Cho and colleagues revealed that loss of NF2 induces a disturbance in the balance of TβR and facilitates TβR1-mediated oncogenic signaling, which could be blocked by TEW7197, a specific TβR1 kinase inhibitor. These findings support TEW7197 would be efficacious drug candidate for NF2-related diseases.Targeting tumor lactate homeostasis is considered to be an attractive approach for cancer therapy. In a cell based screen, Quanz and colleagues have discovered a novel specific and orally available inhibitor (BAY-8002) of the lactate transporter MCT1. BAY-8002 showed potent inhibition of lactate transport in cancer cells and tumor growth inhibition in vivo. The investigation of resistant cells as well as response to treatment in a large cell panel provide new insight into the metabolic plasticity of tumor cells. Furthermore, the results suggest stratification markers to be potentially pursued in the clinical development of MCT1 inhibitors.The receptor tyrosine kinase (RTK) AXL is overexpressed in many human cancers including head and neck squamous cell carcinoma, triple-negative breast cancer, and non-small cell lung cancer. McDaniel, Cummings and colleagues identified a mechanism of resistance to AXL-targeting agents mediated by MERTK, a related RTK. Endogenous MERTK expression increased after treatment with AXL inhibitors and ectopic overexpression conferred resistance to AXL-targeting agents. Moreover, MERTK inhibitors overcame resistance to AXL inhibition and significantly reduced tumor cell expansion and tumor growth. The synergistic interactions between AXL and MERTK inhibitors suggest that therapeutic co-targeting strategies could be very effective in these malignancies.Delivery of therapeutics to pancreatic ductal adenocarcinomas (PDAC) is challenging due to the physical stromal barriers surrounding these tumors. Lo and colleagues applied tumor-penetrating nanocomplexes (TPNs) to breach the stroma and effectively deliver therapeutic siRNA to PDAC. These particles are self-assembled nanostructures comprised of tandem peptides containing both a tumor-penetrating iRGD domain and a cell-penetrating domain, PEGylated derivatives of these tandem peptides, and siRNA. TPNs delivering anti-Kras siRNA exhibited tumor-penetrating ability in 3D organoids and autochthonous tumors, and slowed PDAC growth in vivo. The modular construction of the TPN platform allows for facile adaptation for future genetic targets.

Pharmacokinetic evaluation of the PNC disassembler metarrestin in wild-type and Pdx1-Cre;LSL-KrasG12D/+;Tp53R172H/+ (KPC) mice, a genetically engineered model of pancreatic cancer

PurposeMetarrestin is a first-in-class small molecule clinical candidate capable of disrupting the perinucleolar compartment, a subnuclear structure unique to metastatic cancer cells. This study aims to define the pharmacokinetic (PK) profile of metarrestin and the pharmacokinetic/pharmacodynamic relationship of metarrestin-regulated markers.MethodsPK studies included the administration of single or multiple dose of metarrestin at 3, 10, or 25 mg/kg via intravenous (IV) injection, gavage (PO) or with chow to wild-type C57BL/6 mice and KPC mice bearing autochthonous pancreatic tumors. Metarrestin concentrations were analyzed by UPLC–MS/MS. Pharmacodynamic assays included mRNA expression profiling by RNA-seq and qRT-PCR for KPC mice.ResultsMetarrestin had a moderate plasma clearance of 48 mL/min/kg and a large volume of distribution of 17 L/kg at 3 mg/kg IV in C57BL/6 mice. The oral bioavailability after single-dose (SD) treatment was > 80%. In KPC mice treated with SD 25 mg/kg PO, plasma AUC0–∞ of 14400 ng h/mL, Cmax of 810 ng/mL and half-life (t1/2) of 8.5 h were observed. At 24 h after SD of 25 mg/kg PO, the intratumor concentration of metarrestin was high with a mean value of 6.2 µg/g tissue (or 13 µM), well above the cell-based IC50 of 0.4 µM. At multiple dose (MD) 25 mg/kg/day PO in KPC mice, mean tissue/plasma AUC0–24h ratio for tumor, spleen and liver was 37, 30 and 31, respectively. There was a good linear relationship of dosage to AUC0–24h and C24h. AUC0–24h MD to AUC0–24h SD ratios ranged from two for liver to five for tumor indicating additional accumulation in tumors. Dose-dependent normalization of FOXA1 and FOXO6 mRNA expression was observed in KPC tumors.ConclusionsMetarrestin is an effective therapeutic candidate with a favorable PK profile achieving excellent intratumor tissue levels in a disease with known poor drug delivery.

MTE01.01 GEMM of Lung Cancer

The primary induction of lung cancer is difficult to study in humans because patients often present very late in the course of their disease. Genetically engineered mouse models (GEMMs) have therefore emerged as crucial bridging strategies between understanding pathogenic mechanisms and clinical translation. Importantly, they reveal insights on the events and processes underlying tumor initiation and progression, studies which are not possible when employing transplantation or chemically-induced model systems. The recent advent of next-generation sequencing technologies has provided us with an in-depth characterization of the cancer genome of lung adenocarcinoma (LUAD) (1), squamous cell carcinoma (LUSC) (2) and small cell lung cancer (SCLC) (3). While these studies have highlighted the genetic complexities of lung cancers, attention is now focused on elucidating “driver” mutations that confer a growth advantage, from “passenger” mutations that have little impact on malignant transformation. Investigating the loss or gain-of-function of individual genes, alone or in combination, can be directly addressed using GEMM systems. The “gold-standard” lung cancer models are based on Cre-LoxP recombination technology that enable the formation of autochthonous tumors from a limited number of somatic cells in a spatial and temporal fashion. Critically, tumors arise sporadically within the lung, in the setting of an intact immune microenvironment. GEMMs are designed to harbor genetic mutations frequently identified in human lung cancer. Cre-inducible alleles are engineered to disrupt tumor suppressor genes (LoxP sites flanking key exons (floxed), that are removed upon recombination) and/or activate oncogenes (LoxP-flanked stop codons (lox-stop-lox) that result in gene expression upon recombination). Cre-recombinase is delivered to the lung via inhalation or intra-tracheal injection of a recombinant adenovirus (Ad5) expressing Cre-recombinase under the control of a ubiquitous cytomegalovirus (CMV) promoter. Expression of Cre-recombinase directs the recombination of floxed alleles in a variety of epithelial cell types in the adult mouse lung (4,5). Utilizing this approach enabled investigators to interrogate the functional consequences of genetic alterations found in human lung cancer through the generation of models of LUAD, SCLC and more recently lung LUSC (6). Moreover, the recent advent of CRISPR-Cas9 gene-editing technology now enables us to interrogate the functional interaction between multiple genetic alterations in a high-throughput setting (7). Furthermore, the generation of cell type specific Ad5-Cre viruses, that restrict Cre expression, and thus recombination, to alveolar type II (ATII) (Ad5-SPC-Cre), club (Ad5-CC10-Cre), neuroendocrine (Ad5-CGRP-Cre) and basal (Ad5-K5-Cre, Ad5-K14-Cre) (8) cells, have provided insights into the cellular origins of different subtypes of lung cancer (9,10). Critically, unlike patient-derived xenograft (PDX) models, one additional advantage of GEMMs is the ability to interrogate the interplay between tumor cells and immune cells present in the tumor microenvironment. Such studies are crucial given the success of immune checkpoint inhibitors in lung cancer patients. This presentation will outline lung cancer GEMMs commonly used in the field and how these models can be utilized to identify cancer initiating cells, understand the molecular pathways underlying tumorigenesis, the immune microenvironment of lung cancer, and importantly to identify vulnerabilities that can be exploited for the design of improved treatment modalities for patients. 1. The Cancer Genome Atlas Research Network, Comprehensive molecular profiling of lung adenocarcinoma (2014) Nature, 511 (7511) 543-550. 2. The Cancer Genome Atlas Research Network, Comprehensive genomic characterization of squamous cell lung cancers (2012) Nature, 489 (7417) 519-525. 3. George et al., Comprehensive genomic profiles of small cell lung cancer (2015) Nature, 524 (7563) 47-53. 4. Best et al., Combining cell type-restricted adenoviral targeting with immunostaining and flow cytometry to identify cells-of-origin of lung cancer (2018) Methods in Molecular Biology, 1725 15-29. 5. DuPage et al., Conditional mouse lung cancer models using adenoviral or lentiviral delivery of Cre recombinase (2009) Nature Protocols, 4 (7) 1064-1072. 6. Farago et al., SnapShot: Lung cancer models (2012) Cell, 149 (1) 246-246e1. 7.Rogers et al., A quantitative and multiplexed approach to uncover the fitness landscape of tumor suppression in vivo (2017) Nature Methods, 14 (7) 737-742. 8. Ferone et al., SOX2 is the determining oncogenic switch in promoting lung squamous cell carcinoma from different cells of origin (2016) Cancer Cell, 30 (4) 519-532. 9. Sutherland et al., Cell of origin of small cell lung cancer: inactivation of Trp53 and Rb1 in distinct cell types of adult mouse lung (2011) Cancer Cell, 19 (6) 754-764. 10. Sutherland et al., Multiple cells-of-origin of mutant K-Ras-induced mouse lung adenocarcinoma (2014) Proc. Natl. Acad. Sci. USA, 111 (13) 4952-2957. cell-of-origin, GEMM, tumor development

Abstract PR06: Investigating ectopic lymphoid aggregates in a genetically engineered mouse model of lung adenocarcinoma

Abstract Ectopic lymphoid aggregates (LA) located in the vicinity of solid tumors have been identified in human patients with many types of cancers and have been largely associated with improved outcomes. In particular, LA in patients with non-small cell lung cancer have been associated with longer overall survival, regardless of the presence of intratumoral CD8 T cells, a commonly used prognostic marker. Tumor-associated LA are organized similarly to lymph nodes with distinctive T-cell zones, B-cell zones, and high endothelial venules (HEV), suggesting a role of LA in the regulation of an antitumor immune response. Therefore, therapeutics targeting these LA have potential to strengthen the antitumor immune response and consequently improve patient outcome. Nevertheless, there is an absence of preclinical models that mirror this clinically relevant phenomenon, and thus their formation and precise function(s) remains poorly understood. Using a genetically engineered mouse model (GEMM) of lung adenocarcinoma in immunocompetent KP-T mice (KrasLox-STOP-Lox-G12D;p53flox/flox;Rosa26Lox-STOP-Lox-tdTomato), we have observed that tumor-associated LA develop and serve as critical sites for the regulation of antitumor immune responses. In this model, autochthonous tumors develop about 20 weeks after intratracheal infection with Cre recombinase-encoding lentivirus (Cre LV), and progress in a similar manner to human disease. The expression of an antigenic construct containing three well-characterized T-cell antigens fused to luciferase (LucOS) was introduced into developing tumors via LV (LucOS/Cre LV). Interestingly, tumors expressing these antigens are physically associated with LA, whereas tumors that do not express these antigens are not. We are therefore uniquely positioned to investigate the role of tumor antigens, as well as the subsequent antitumor immune response, in the development of tumor-associated ectopic LA. We hypothesize that tumor antigen-specific, “pioneering” T cells trigger the recruitment of other immune cell types and ultimately lead to the formation of tumor-associated LA and the development of an antitumor immune response. Future studies will investigate the role of tumor-specific CD8 T cells in the formation of LA, using various GEMM and LV infections to manipulate and “turn off” the expression of T cell antigens by tumor cells. To advance our understanding of cancer outcomes and select more effective, individualized treatments for patients, we believe that it is crucial to directly interrogate how and when LA develop. Ultimately, our findings will identify cellular and molecular targets essential to the formation and maintenance of LA, thus providing candidates for therapeutic modulation of these clinically relevant tumor-associated immune structures. This abstract is also being presented as Poster B14. Citation Format: Kelli Connolly, Mursal Nader, Nikhil Joshi. Investigating ectopic lymphoid aggregates in a genetically engineered mouse model of lung adenocarcinoma [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr PR06.