RESEARCH
Lung cancer is the most common cause of cancer-related-death world-wide. About 85% of lung cancers are non-small cell lung cancer (NSCLC), mostly identified after metastatic disease has evolved and are thus incurable. Significant advances have been made in recent years in the identification of driver mutations whose inhibition can prolong survival. However, metastatic spread and development of resistance to therapy is common, and overall five-year survival rates for lung cancer patients are around 15%. In order to improve this dismal outcome, deeper understanding into the molecular mechanisms occurs in the tumor and in its microenvironment is required and is the main goal of our research.
Tumor microenvironment
Tumor-host interactions play a major role in malignancies’ initiation and progression . We have reported in the past that tumor cells attenuate stress-induced p53 activation in neighboring cancer cells (Bar et al., Cancer cells suppress p53 in adjacent fibroblasts, Oncogene. 28 (2009). doi:10.1038/onc.2008.445). We have further investigated the impact of cancer cells on the p53-pathway in lung cancer stroma. We have used primary cancer-associated fibroblasts (CAFs) grown from resected human lung cancers and models of Lung cancer lines as well as ex-vivo organ cultures. We found that conditioned medium (CM) of lung cancer cells rapidly induce protein levels of stromal-Mdm2. CM of fibroblasts had no such effect. Mdm2 induction occurred through enhanced translation, was mTORC1-dependent and correlated with activation of AKT and p70 S6K. Mdm2 knockdown in fibroblasts reduced the invasion of neighboring cancer cells, independently of stromal-p53. Mdm2 overexpression in fibroblasts enhanced the invasion of cancer cells and the growth of inoculated tumors in mice. Our results indicate stromal-Mdm2 participates in a major cancer-host feedback mechanism. Soluble cancer-originated signals induce enhanced translation of stromal-Mdm2 through mTORC1 signaling, which in turn enhances the invasion ability of neighboring cancer cells (Kamer et al. “Stromal-MDM2 Promotes Lung Cancer Cell Invasion through Tumor-Host Feedback Signaling.” Molecular cancer research : MCR vol. 18,6 (2020): 926-937. doi:10.1158/1541-7786.MCR-19-0395.).
Our study suggests a critical role of stromal MDM2 elevation in cancer cell invasion, and a dispensable role of stromal p53. Cancer cells secrete factors that enhance MDM2 protein levels in adjacent stromal-fibroblasts. Stromal-MDM2 in turn enhances cancer cell invasion in a p53-independent manner.
Brain metastasis in NSCLC
Non-small cell lung cancer (NSCLC) is the most common cause of cancer-death due to early metastatic spread, in many cases primarily to the brain. Organ-specific pattern of spread of disease might be driven by the activity of a specific signaling pathway within the primary tumors. Presently, we are lacking tools to predict which LC patient with a primary resectable LC will develop brain metastases (BM). Identification of such patients could enable more frequent monitoring and thereby detection of BM at an earlier stage, resulting in more efficient treatment and improved survival. There are two projects investigating brain mets spread conducted in our lab:
1) We have identified an expression signature of genes with the development of brain metastasis (BM) after surgical resection of NSCLC. We have used rapidly frozen NSCLC surgical specimens and analyzed them by RNA-sequencing (Illumina HiSeq 2500). Clinical parameters and gene expression were examined for differentiating between patients with BM, patients with metastases to sites other than brain, and patients who did not develop metastatic disease at a clinically significant follow up. Principal component analysis and pathway enrichments studies were done. A total of 91 patients were included in this study, 32 of which developed BM. Stage of disease at diagnosis (P=0.004) and level of differentiation (P=0.007) were significantly different between BM and control group. We have identified a set of 22 genes which correlated specifically with BM, and not with metastasis to other sites. This set achieved 93.4% accuracy (95% CI: 86.2–97.5%), 96.6% specificity and 87.5% sensitivity of correctly identifying BM patients in a leave-one-out internal validation analysis. A significant pathway that was identified and strongly correlated with BM risk was the oxidative phosphorylation pathway (Kamer et al. “Predicting brain metastasis in early stage non-small cell lung cancer patients by gene expression profiling.” Translational lung cancer research vol. 9,3 (2020): 682-692. doi:10.21037/tlcr-19-477).
Oxidative phosphorylation pathway genes are upregulated in BM patient specimens. (A) PCA plot of all patients included in
this study color-coded by brain metastasis development (black/red Control/BM respectively). (B) Boxplots of genes from the Oxidative
Phosphorylation pathway that were differentially expressed between BM and Control patients. *, P<0.05; **, P<0.005. n.s., non-significant.
2) Predicting brain metastasis in cancer by exosome analytics - Tumor-derived small extracellular vesicles (sEVs) from tumors released into blood and have gained huge interest as a tool that may correlate with different clinical outcome parameters. Moreover, it has been demonstrated that sEVs foster a pre-metastatic niche in multiple tumor types. sEVs released into blood are partly a mirror of their tumor of origin and therefore offer a good source for RNA as well as proteins for biomarker analyses. In this project, we focus on NSCLC tumor-released sEVs and aim to identify RNA and protein signatures that are specific to NSCLC patients that will develop BM.
For that propose, we are using plasma samples from a retrospective cohort consists of three subgroups: patients that developed BM within three years after surgery as the first metastatic site (BM group); patients that developed metastatic disease but not BM as the first site within this time frame and patients that did not develop any metastatic disease in at least 3-year follow-up. Exosomes are isolated using IZON columns and will be used to profile proteins and RNA for each of the groups. We have collected so far hundreds of samples with full clinical data and the project is currently in his first steps.
This research is supported by The Ministry of Science and Technology of Israel - Research program Lise Meitner Grants for Israeli-Swedish research collaboration, with collaboration with Irit Gat-Viks from TAU and Rolf Lewensohn from Karolinska Institute.
Translational Research
Using samples from clinical studies led by Prof. Bar, as well as tumor samples and blood collected from NSCLC patients during routine resections, we aim to understand the mechanisms of response to immune checkpoint inhibitors being used as a first line treatment in lung cancer.
Ongoing studies:
1) Dissecting the role of B-cells in lung cancer response to checkpoint inhibitors -
The role of B-cells is currently evaluated in our lab using samples from investigator-initiated study of neo-adjuvant anti-PD-1 (pembrolizumab) for early NSCLC (A). We have identified B-cells as the cellular component most responsive to CPI treatments and mostly correlated with it’s efficacy. Needle biopsies taken from participants prior- and post-treatment were analyzed by nanostring GeoMx Digital Spatial Profiler, allowing assessment of protein and mRNA expression in regions of interest. CD20 protein was found to be the most significantly differently expressed in the responding vs. the non-responding patients (B), as was TLS density. In addition, CD20 was the most induced protein following anti-PD-1 treatment in responders, similarly to data recently reported for melanoma. Multiplex ion-beam imaging (MIBI) analysis done by our collaborator, Dr. Leeat Keren from the Weizmann institute, of the post-CPI surgical samples corroborated the importance of B-cells, and identified plasma cells as most correlated with response. These results provide further evidence for the importance of B-cells in CPI-response of NSCLC.
2) Longitudinal profiling of PBMCs’ T-cell repertoire to assess long-term immunologic tumor control following neoadjuvant pembrolizumab for early NSCLC - The goal of the study is to evaluate a potential correlation between the characteristics of the T-cell clones of patients undergoing neoadjuvant pembrolizumab treatment and the clinical outcome of these patients. The samples analyzed in this study include tumor samples collected at surgery following neo-adjuvant pembrolizumab treatment (as part of a clinical study; fresh frozen), blood samples collected before initiation of the study treatment and after the completion of the treatment (at time of the surgery). In addition we use blood samples collected during follow-up of the patients.
All 138 samples were sent to Prof. Efroni lab (Bar-Ilan University) for T cell repertoire sequencing.
Sample collection during the study. A. All samples that were collected during the study for each patient. Pre – pre-treatment blood sample, Post – post-treatment blood sample. Tumor – tumor sample from surgery. Follow up – blood samples taken post-surgery in the years indicated in the upper bar. Total number of blood samples in the study – n=138. Of these, 84 were obtained in the follow-up phase. B. Out of 20 patients, 7 responded to the neoadjuvant treatment (R-responders) and 13 didn’t respond (NR – non responders). Two patients from the NR group had recurrence of the disease. No recurrence was evident for patients that responded to treatment to this end.
3) Ex-vivo models to study immunotherapy - One of the major challenges for the advancement of cancer immunotherapy is lack of robust, accessible experimental models. We have established an ex-vivo organ culture (EVOC) model of immunotherapy for non-small cell lung cancer (NSCLC). We used either freshly resected early stage tumors that are collected from the operating room or pleural effusions drained from metastatic lung cancer patients.
A. Early stage-lung cancer EVOC model – Freshly resected lung tumors are used to produce EVOCs as a model to evaluate the response to anti-PD-L1, anti-CTLA-4, and their combination. The response to immune checkpoint inhibitors (ICI) was assessed by IFNγ gene induction and secretion. Tumors are dissociated to clusters of epithelial cells and immune single cells. The cells are cultured with fetal calf serum and human autologous serum. We found that EVOCs maintained tumor microenvironment and 3D features and were found to be in vitro responsive to ICI treatments. EVOCs were treated either with single ICI or combinations. The cohort demonstrated similar characteristics to the real-world data of NSCLC response to ICI. The overall response rate was 23%, in accordance to the results in unselected NSCLC patients. All tumors of responders-EVOCs had "hot tumor" characteristics, including higher presence of CD4+ and CD8+ cells, and higher PD-L1 expression. Retention of the microenvironment and minimal addition of exogenous factors suggest this model to reliably represent the original tumor. The cluster-based EVOC model we describe can provide a valuable, yet simple and widely applicable tool for the study of immunotherapy in NSCLC (Kamer et al. “Immunotherapy response modeling by ex-vivo organ culture for lung cancer.” Cancer immunology, immunotherapy : CII vol. 70,8 (2021): 2223-2234. doi:10.1007/s00262-020-02828-w.).
EVOC preparation -Tumor tissue is collected directly from the operating room, rapidly dissected with surgical razor blades, followed by gentleMACS dissociation. Released cells and clusters are washed and cultured in medium supplemented with autologous serum and fetal calf serum. Drugs are added to the culture medium and culture is incubated for four days.
IHC of a representative EVOC; upper panel—microscopic images of 10 × magnification, lower panels—enlargement of the cluster area. IHC of CK7 (left panel), CD8 (middle panel) and PD-L1 (right panel)
Heat map of 12 proteins expression in immune-responsive and non-responsive EVOCs with hierarchical clustering. Rows correspond to IHC quantification of individual proteins, columns correspond to EVOC samples. Besides the proteins depicted in A, IHC results for CD21 and CD23, identifying follicular dendritic cells, and KI67, indicative of cell proliferation rate, were also included. IHC of PD-L1, quantified separately for immune
and tumor cells was incorporated. At the bottom: immunotherapy
response of the corresponding EVOCs, coded as indicated. H&E: Hematoxylin and eosin. Granz.B: granzyme B
B. Ex-vivo culture from lung cancer pleural effusions - a model for checkpoint inhibitors response
Pleural effusions (PE) are accumulations of excessive fluids in the pleural space that surrounds each lung. PEs appears in rough ly 20 % of advanced lung cancer patients and its presence is associated with worse prognosis. Project aim : Establish ex vivo primary culture from PEs of metastatic NSCLC patients as a tool to investigate and predict response to immune checkpoint inhibitors (ICI). Methods : PE samples were collected following clinically indicated drainage from advanced NSCLC patients at the Sheba pulmonary department and at the onco logy department. Cell populations present in the fluids were characterized by FACS and FFPE cell blocks. An ex vivo culture model (EV OC) was established to test and study response to ICI. Results : Fresh PEs collected ( 31 ) were found to include mostly lymphocytes (CD 4 + and some CD 8 + cells), as well changing percentages of cancer epithelial cells. Out of 10 EVOCs that were treated with ICI, six ( 60 %) exhibited significant induction of IFN g gene and secretion of IFN g protein. Two sequential samples from the same patient were tested, demonstrating in one sample no response (taken shortly after disease progression on ICI), while a later sample did respond to ICI ex vivo. Analysis o f these samples demonstrated elevated CD 20 + cells, PDL 1 upregulation and reduced FoxP 3 + cells in the responding sample. Conclusion : EVOCs of pleural effusions may provide insight into the immune response to ICIs. The identification and characterization of tumors that response may help to tailor the appropriate treatment to each patient, and hence to provide better therapeutic effect.
Cartoon and X-Ray images demonstrating the accumulation of pleural effusion in the pleural space.
Representative IHC staining of PE cell block is presented. Antibodies for EpCAM (epithelial cells), CD4 (T helper and T regs), CD8 (cytotoxic T cells) and PD-L1 were used. Epithelial cells appear in clusters or as single cells and varies in percentage in different patients. CD4 lymphocytes are more abundant ( 50-70%) than CD8 cells (10-30%) in most of PE samples. Correlation to response will be determined. Scale bar in the left images apply to all images on the same row.