Ceramide metabolism and intrinsic radiation response of NSCLC tumors
In 2013, our group got awarded with a position called «Delt forskerstilling» from Helse-Nord, which comprised 50% research time during 3 years for 2 candidates; MTG & TH. Hence, two different but related projects were appointed to the research group: A) “Ceramide metabolism and intrinsic radiation response of NSCLC tumors” (in vitro) and B) “Role of tumor stromal fibroblasts in the overall response of NSCLC tumors to radiotherapy” (in vivo). This report represents the “end-of-project report” corresponding to project (A), in which TH was the applicant. BACKGROUND: The ultimate goal of our research efforts is to improve radio-curability of treatment-resistant lung cancers, effectuated by exploring responses of different tumor stromal cells to standard low-dose as well as high-dose radiation therapy regimens. In this project, the original plan was to extend our scientific efforts towards ceramide-metabolism and its influence on the intrinsic radio-responses of lung tumors. However, initial experiments performed by the group prompted us to focus our strategy towards radiation effects on the immune-regulatory mechanisms in tumors instead. The new focus has since then been to explore the role of CAFs as regulators of tumor immunity, and the potential disturbances of this function by radiation. NEW AIMS: Explore immune-regulatory functions exerted by Carcinoma-Associated Fibroblasts (CAFs) in lung tumors. Sub-aims: A) Explore immune-regulatory functions by CAFs in VITRO (i.e. in primary cell cultures) B) Explore immune-regulatory functions by CAFs in VIVO (i.e. by xenograft tumors in animals) C) Explore immune-regulatory functions by CAFs in SITU (human tumor tissues). RESULTS & CONCLUSIONS: Aim A) From our recent studies IN VITRO, three main conclusions can be highlighted: i) CAFs display a powerful immunosuppressive effect over allogenic T-cells; ii) High-dose radiotherapy does not alter the immunosuppressive functions of CAFs; iii) High-dose radiotherapy does not induce immunogenic cell death responses in CAFs (Gorchs et al. 2015). Aim B) In the IN VIVO study, we have demonstrated that irradiated CAFs lose their pro-tumorigenic effects when co-implanted in xenograft tumors. This finding represents a previously unknown advantageous (and indirect) effect induced by radiotherapy (Grinde et al. 2017 submitted to Scientific Reports). Aim C) Recent work IN SITU is based on tissue micro-arrays (TMAs) on human lung tumor specimens, and we have found that CAF abundance correlates poorly with the extent of tumor-infiltrating inflammatory or immune cells, however, the presence of FAP1+ CAFs associates with better prognosis in tumors infiltrated by effector T-cells (Kilvær et al. 2017 in prep.). Scientific productivity/DISSEMINATION: Our work has been orally presented at the following events: A) National Videoconference, University Hospital of Northern Norway Feb24 2013; B) National meeting Mini-MedFys 2014. Trondheim, Norway. March 3 2014. C) Intl Work-shop: “Immune and stromal responses in cancerology: New challenges for therapeutic targeting”. Nantes, France. Oct-1-4 2014; D) 9th Intl Conference of Anticancer Research. Sithonia, Greece, Oct 4-7 2014. E) Nordic PhD Course. UiT, April 16 2015. F) Cell Symposia, Sitges, Spain. June 14-16 2015. G) International conference on Cancer-associated Fibroblasts. Bergen, Norway. Oct 22-23 2016. H) International symposium “Small animal precision image-guided radiotherapy”. Ghent March 21-23 2016. Planned presentations: A) Annual meeting AACR (American Association for Cancer Research) Washington DC, USA. April 1-5 2017. B) EACR-AACR-SIC 2017: June 24-27 Florence, Italy. PUBLICATIONS: During the granted period, we have published four original manuscripts and one review in international peer reviewed journals. Additionally, we currently have one submitted manuscript and three manuscripts in preparation.
Accumulating evidence propose crucial roles of the tumor stroma in the responses of cancers to therapy. However, the role of tumor fibroblasts (CAFs) on radiotherapy and immunotherapy outcomes remain poorly characterized. A better understanding of the interplay between CAFs and immune cells and tumor cells will aid in therapeutic improvements. Aim A: In vitro studies Outcomes of the study: CAFs exert powerful immunosuppressive effects on T-lymphocytes in vitro via the secretion of paracrine soluble factors. Additionally, the CAF-mediated immunosuppressive effects remain unchanged after ionizing radiation. Clinical relevance: These results suggest that CAFs are immunosuppressive and may exert counterbalancing effects in an immunotherapy context. Approaches for blocking CAF-derived immunosuppressive signals may enhance the efficacy of cancer immunotherapies. Aim B: Pre-clinical animal study Outcomes of the study: CAFs lose their pro-tumorigenic effects after exposure to ionizing radiation. Tumor infiltration by inflammatory cells remain similar in tumors established with and without CAFs. Clinical relevance: The results uncover a previously unknown positive effect elicited by ionizing radiation. This study suggest the use of high-dose radiotherapy in lung tumors with high degree of tumor stroma. Aim C: In situ tumor tissue study Outcomes of the study: CAF abundance in tumor tissues do not correlate with infiltration of tumors by immune cells, however, FAP1+ CAFs correlates with better prognosis in patients with high immune infiltrated tumors. Clinical relevance: The study suggest that a subset of CAFs exert immuno-adjuvant effects, and thus strategies to selectively target CAFs for the improvement of immunotherapy should be cautiously considered.
Ceramide metabolism and intrinsic radiation responses of NSCLC tumors
Radiotherapy outcome depends on the total response from tumor and stromal cells, but stromal effects from radiation remains poorly defined. Our project aims at exploring biological responses of radiation in lung tumor stroma, for ultimately uncovering new targets for pharmacological manipulation towards enhanced radioresponses and patient survival.
BACKGROUND & SIGNIFICANCE: Cancer-associated fibroblasts (CAFs) are a heterogeneous group of cells found in large numbers inside and around solid tumors. In many cancer types, high abundance of intra-tumoral CAFs is associated with poor prognosis, and their role as important determinants of tumor establishment and progression is widely acknowledged. In the context of radiotherapy (RT), all tumor-associated host cells within the planning target volume will receive the same prescribed radiation dose as malignant cells. Radioresponses by the tumor microenvironment is therefore ultimately affecting the overall therapeutic outcome. Given the fundamental role played by CAFs on general tumor growth regulation, we hypothesize that CAFs may change their phenotype and pro-malignant nature upon irradiation, and that this circumstance could influence the ultimate fate of tumors and patients post-therapy. AIMS: During the last year, we have been exploring a) whether irradiation alters the pro-malignant influence of CAFs in vivo; b) if diverse dose/fraction regimens exert different CAF-mediated tumor growth regulations; and c) potential mechanism(s) behind the radiation-induced effects, by histopathology analyses of tumors. METHODOLOGY: Along with lung tumor cells, Irradiated and non-irradiated CAFs have been implanted into immuno-compromised mice. Tumor growth kinetics have been monitored and tumor biological parameters including vessel density, tumor cell proliferation, collagen deposition and immune cell infiltration have been analyzed on tumor tissue specimens and quantified manually or by software. RESULTS/CONCLUSIONS: Our findings indicate that co-administration of CAFs with lung tumor cells increases growth of xenograft tumors, but irradiated CAFs lose this pro-tumorigenic capacity. Histopathology assessments of major tumor biological features do not reveal any clear mechanism behind the observed effects, and CAF-mediated pro-tumorigenic effects apparently occur at early stages of tumor-initiation. To finalize this work, we are currently investigating the extent of tumor infiltration by natural killer (immune) cells as well as CAF-behavior post-transplantation. SCIENTIFIC Productivity/Dissemination: During the year 2015, our group has presented scientific results at the following meetings: 1) CELL symposia: Cancer, Inflammation and Immunity, June 14-16, 2015; Sitges, Spain (Poster). 2) International meeting on Cancer-Associated Fibroblasts. Bergen, Norway; October 22-23 2015 (Oral+Poster). Additionally, two original international peer review articles have been published in 2015; a) CAF-mediated immuno-suppressive effects and b) Prognostic impact of CAF-related molecular markers in human tissue specimens. Recent results generated in animals will be presented at MedFys-2016 (Kvitfjell, Feb 8-10)(Oral+poster) and at the international meeting “3rd Symposium on Small Animal Precision Image-Guided RadioTherapy” (Ghent, Belgium, 21-23 March 2016) (Oral).
Ceramide-metabolism and the intrinsic radiation responses of NSCLC tumors
During radiotherapy, both malignant and non-malignant tumor cells are exposed to ionizing radiation, but radiation effects on tumor stroma remain poorly studied. Our project aims at widening our understanding on biological responses of lung tumor stroma to radiation, to ultimately achieve enhanced local tumor control and overall patient survival.
Aim & Milestone plan: The ultimate goal of our research efforts is to improve radio-curability of treatment-resistant lung cancers, by exploring the responses of the different tumor components to standard low-dose as well as high-dose radiation regimens. In this project, the original plan was to investigate ceramide-metabolism and its influence on the intrinsic radio-responses of lung tumors. However, initial experiments performed by the group led us to shift the angle of our strategy towards radiation effects on the immune-regulatory mechanisms in tumors. The new focus has since then been to explore the role of CAFs as regulators of tumor immunity, and the potential disturbances of this function by radiation. Methodology: Primary CAF-cultures have been established from lung cancer specimens derived from patients operated for non-small cell lung cancer. Irradiated and non-irradiated CAFs were examined for immune-modulation in experiments with peripheral blood mononuclear cells. Regulation of lymphocytes behavior was checked by proliferation assay, migration assay and cytokine expression assay. Additionally, CAF-secreted immune-regulatory factors were studied by multiplex protein arrays, ELISAs and LC-MS/MS proteomics. Immunogenic cell death responses in CAFs were studied by analyzing the release of HMGB1 and ATP. Results/Conclusions: Three main conclusions can be highlighted from our study: i) CAFs display a powerful immuno-suppressive effect over T-cells; ii) High-dose radiotherapy does not alter the immuno-suppressive functions of CAFs; iii) High-dose radiotherapy does not induce immunogenic cell death responses in CAFs. Scientific productivity/Dissemination: This work has been orally presented at i) International Work-shop: “Immune and stromal responses in cancerology: New challenges for therapeutic targeting”. Nantes, France. Oct-1-4 2014; and ii) 9th Intl Conference of Anticancer Research. Sithonia, Greece, Oct 4-7 2014. Additionally, an original article has recently been submitted to the journal “Frontiers in Oncology”, and constitutes our contribution to the Research Topic “Radiobiological clues towards optimizing high-dose radiotherapy” launched by us in the same journal.
Ceramide metabolism and intrinsic radiation response of NSCLC tumors
Trass det faktum at stråleterapi affiserer både tumorceller og omkringliggende vev, er effekter av stråling på stromalt vev lite utforsket. Våre studier tar sikte på å øke forståelsen for resulterende gunstige og ugunstige biologiske responser etter stråleterapi. Ervervet kunnskap vil bidra til økt behandlingsrespons og dermed økt overlevelse.
Stråleterapi har lenge utgjort en av hjørnesteinene i kreftbehandlingen, noe som reflekteres i det faktum at over 50 % av alle kreftpasienter mottar strålebehandling i løpet av sin sykdomsperiode. Videre er det nye konseptet høy-dose stråleterapi på full fart inn i klinikkene, og har introdusert et paradigmeskifte i forhold til behandlingsstrategi og prognoser for pasienter med lungekreft. Vårt translasjonelle forskningsprosjekt fokuserer på strålerelaterte responser i det tumor-assosierte mikromiljøet, og anvender lungekreft som tumormodell og utgangsmateriale for biologiske studier. Våre innledende forskningsresultater har spesifikt analysert stråleresponsen hos tumor-assosierte fibroblaster, som er en meget stråleresistent celletype. Prosjektet ”Ceramide-metabolism and the intrinsic radiation responses of NSCLC tumors”; HN-ID 9225/SFP 1138-13) representerer en videreutvikling av våre etablerte strålebiologiske studier på cellekulturer. Parallelt med disse studiene har vi også igangsatt et prosjekt der vi viderefører våre studier i eksperimentelle dyremodeller (”Role of tumor stromal fibroblasts in the overall response of NSCLC tumors to radiotherapy”; HN-ID 9086/SFP1137-13). Min stilling som forsker (50 %) på prosjektet ”Ceramide-metabolism and the intrinsic radiation responses of NSCLC tumors” ble aktivert 01.05.13. Som overordnet prosjektleder omfatter min aktivitetsplan også arbeid ift etablering av prosjektrelaterte dyremodeller (”Role of tumor stromal fibroblasts in the overall response of NSCLC tumors to radiotherapy”). Således har jeg i den inneværende prosjektperioden hatt fokus på følgende arbeidsområder:
1) Innhenting av bakgrunnslitteratur og skriving av oversiktsartikkel.
Artikkelen er nå publisert i journalen ”Frontiers in Oncology” (2014, vol. 4 (1), 1-12).
2) Pilotforsøk ift mekanismer rundt celledød/ celleoverlevelse etter bestråling av cellekulturer. Vi har utforsket mekanistiske forskjeller rundt strålesensitive og stråleresistente cellelinjer, og informasjonen vil anvendes for videre studier i vårt eksperimentelle system. Innsamlede data rundt disse forsøkene er for tiden under evaluering, og undertegnede vil i løpet av inneværende semester ferdigstille dette arbeidet. Første utkast til manuskript er forventet å foreligge per medio 2014.
3) Etablering av preklinisk (smådyrs) PET-CT avbildning samt anbudsprosess rundt innkjøp av preklinisk (smådyrs) stråleenhet. Som et ledd i dette arbeidet har vi hatt to åpne forelesninger fra to ulike leverandører av preklinisk stråleenhet (X-RAD og X-Strahl). Videre foretok vi i november (6.-11.) en befaring til Manchester (Wolfson Molecular Imaging Centre) og Glasgow (The Beatson Cancer Institute) for å se dette utstyret i bruk, samt for diskusjon rundt fremtidige samarbeidsprosjekter.
Cancer Associated Fibroblasts in Stage I-IIIA NSCLC: Prognostic Impact and Their Correlations with Tumor Molecular Markers.
PLoS One 2015;10(8):e0134965. Epub 2015 aug 7
Cancer-associated fibroblasts from lung tumors maintain their immunosuppressive abilities after high-dose irradiation.
Front Oncol 2015;5():87. Epub 2015 mai 12
Radiotherapy and the tumor stroma: the importance of dose and fractionation.
Front Oncol 2014;4():1. Epub 2014 jan 21
Tumorigenic responses of Cancer-Associated stromal Fibroblasts after ablative radiotherapy: A transcriptome-profiling study.
Journal of Cancer Therapy.
Ionizing radiation abrogates the pro-tumorigenic capacity of Cancer-Associated Fibroblasts co-implanted in xenografts.
Radiotherapy and the tumor stroma: the importance of dose and fractionation
Frontiers in ONCOLOGY, 21.01.14, vol4., article1, p 1-12. doi: 10.3389/fonc.2014.00001