Inigo Martinez-Zubiaurre

Radiotherapy and the Tumor Stroma: The Importance of Dose and Fractionation

Ionizing radiation is a non-specific but highly effective way to kill malignant cells. However, tumor recurrence sustained by a minor fraction of surviving tumor cells is a commonplace phenomenon caused by the activation of both cancer cell intrinsic resistance mechanisms, and also extrinsic intermediaries of therapy resistance, represented by non-malignant cells and structural components of the tumor stroma. The improved accuracy offered by advanced radiotherapy (RT)-technology permits reduced volume of healthy tissue in the irradiated field, and has been triggering an increase in the prescription of high-dose oligo-fractionated regimens in the clinics. Given the remarkable clinical success of high-dose RT and the current therapeutic shift occurring in the field, in this review we revise the existing knowledge on the effects that different radiation regimens exert on the different compartments of the tumor microenvironment, and highlight the importance of anti-tumor immunity and other tumor cell extrinsic mechanisms influencing therapeutic responses to high-dose radiation.

Cancer-associated fibroblasts from human NSCLC survive ablative doses of radiation but their invasive capacity is reduced

BackgroundCancer-Associated Fibroblasts (CAFs) are significant components of solid malignancies and play central roles in cancer sustainability, invasion and metastasis. In this study we have investigated the invasive capacity and matrix remodelling properties of human lung CAFs after exposure to ablative doses of ionizing radiation (AIR), equivalent to single fractions delivered by stereotactic ablative radiotherapy (SART) for medically inoperable stage-I/II non-small-cell lung cancers.MethodsCAFs were isolated from lung tumour specimens from 16 donors. Initially, intrinsic radiosensitivity was evaluated by checking viability and extent of DNA-damage response (DDR) at different radiation doses. The migrative and invasive capacities of CAFs were thereafter determined after a sub-lethal single radiation dose of 18 Gy. To ascertain the mechanisms behind the altered invasive capacity of cells, expression of matrix metalloproteinases (MMPs) and their endogenous inhibitors (TIMPs) were measured in the conditioned media several days post-irradiation, along with expression of cell surface integrins and dynamics of focal contacts by vinculin-staining.ResultsExposing CAFs to 1 × 18 Gy resulted in a potent induction of multiple nuclear DDR foci (> 9/cell) with little resolution after 120 h, induced premature cellular senescence and inhibition of the proliferative, migrative and invasive capacity. AIR promoted MMP-3 and inhibited MMP-1 appearance to some extent, but did not affect expression of other major MMPs. Furthermore, surface expression of integrins α2, β1 and α5 was consistently enhanced, and a dramatic augmentation and redistribution of focal contacts was observed.ConclusionsOur data indicate that ablative doses of radiation exert advantageous inhibitory effects on the proliferative, migratory and invasive capacity of lung CAFs. The reduced motility of irradiated CAFs might be a consequence of stabilized focal contacts via integrins.

Cancer-associated fibroblasts from lung tumors maintain their immunosuppressive abilities after high-dose irradiation.

Accumulating evidence supports the notion that high-dose (>5 Gy) radiotherapy (RT) regimens are triggering stronger pro-immunogenic effects than standard low-dose (2 Gy) regimens. However, the effects of RT on certain immunoregulatory elements in tumors remain unexplored. In this study, we have investigated the effects of high-dose radiotherapy (HD-RT) on the immunomodulating functions of cancer-associated fibroblasts (CAFs). Primary CAF cultures were established from lung cancer specimens derived from patients diagnosed for non-small cell lung cancer. Irradiated and non-irradiated CAFs were examined for immunomodulation in experiments with peripheral blood mononuclear cells from random, healthy donors. Regulation of lymphocytes behavior was checked by lymphocyte proliferation assays, lymphocyte migration assays, and T-cell cytokine production. Additionally, CAF-secreted immunoregulatory factors were studied by multiplex protein arrays, ELISAs, and by LC-MS/MS proteomics. In all functional assays, we observed a powerful immunosuppressive effect exerted by CAF-conditioned medium on activated T-cells (p > 0.001), and this effect was sustained after a single radiation dose of 18 Gy. Relevant immunosuppressive molecules such as prostaglandin E2, interleukin-6, and -10, or transforming growth factor-β were found in CAF-conditioned medium, but their secretion was unchanged after irradiation. Finally, immunogenic cell death responses in CAFs were studied by exploring the release of high motility group box-1 and ATP. Both alarmins remained undetectable before and after irradiation. In conclusion, CAFs play a powerful immunosuppressive effect over activated T-cells, and this effect remains unchanged after HD-RT. Importantly, CAFs do not switch on immunogenic cell death responses after exposure to HD-RT.

Ionizing radiation abrogates the pro-tumorigenic capacity of cancer-associated fibroblasts co-implanted in xenografts

Cancer-associated fibroblasts (CAFs) are abundantly present in solid tumors and affect tumorigenesis and therapeutic responses. In the context of clinical radiotherapy, the impact of irradiated CAFs to treatment outcomes is largely unexplored. Aiming at improving radiotherapy efficacy, we have here explored the effect of radiation on the inherent pro-tumorigenic capacity of CAFs in animals. Ionizing radiation was delivered to cultured CAFs as single-high or fractionated doses. Tumor development was compared in mice receiving A549 lung tumor cells admixed with irradiated or control CAFs. Biological mechanisms behind tumor growth regulation were investigated by quantitative histology and immunohistochemistry. Viability assessments confirmed that irradiated CAFs are fully functional prior to implantation. However, the enhanced tumorigenic effect observed in tumors co-implanted with control CAFs was abrogated in tumors established with irradiated CAFs. Experiments to ascertain fate of implanted fibroblasts showed that exogenously administered CAFs reside at the implantation site for few days, suggesting that tumor growth regulation from admixed CAFs take place during initial tumor formation. Our work demonstrate that irradiated CAFs lose their pro-tumorigenic potential in vivo, affecting angiogenesis and tumor engraftment. This finding propose a previously unknown advantageous effect induced by radiotherapy, adding to the direct cytotoxic effects on transformed epithelial cells.

Cleavage of the urokinase receptor (uPAR) on oral cancer cells: regulation by transforming growth factor – β1 (TGF-β1) and potential effects on migration and invasion

BackgroundUrokinase plasminogen activator (uPA) receptor (uPAR) is up-regulated at the invasive tumour front of human oral squamous cell carcinoma (OSCC), indicating a role for uPAR in tumour progression. We previously observed elevated expression of uPAR at the tumour-stroma interface in a mouse model for OSCC, which was associated with increased proteolytic activity. The tumour microenvironment regulated uPAR expression, as well as its glycosylation and cleavage. Both full-length- and cleaved uPAR (uPAR (II-III)) are involved in highly regulated processes such as cell signalling, proliferation, migration, stem cell mobilization and invasion. The aim of the current study was to analyse tumour associated factors and their effect on uPAR cleavage, and the potential implications for cell proliferation, migration and invasion.MethodsMouse uPAR was stably overexpressed in the mouse OSCC cell line AT84. The ratio of full-length versus cleaved uPAR as analysed by Western blotting and its regulation was assessed by addition of different protease inhibitors and transforming growth factor - β1 (TGF-β1). The role of uPAR cleavage in cell proliferation and migration was analysed using real-time cell analysis and invasion was assessed using the myoma invasion model.ResultsWe found that when uPAR was overexpressed a proportion of the receptor was cleaved, thus the cells presented both full-length uPAR and uPAR (II-III). Cleavage was mainly performed by serine proteases and urokinase plasminogen activator (uPA) in particular. When the OSCC cells were stimulated with TGF-β1, the production of the uPA inhibitor PAI-1 was increased, resulting in a reduction of uPAR cleavage. By inhibiting cleavage of uPAR, cell migration was reduced, and by inhibiting uPA activity, invasion was reduced. We could also show that medium containing soluble uPAR (suPAR), and cleaved soluble uPAR (suPAR (II-III)), induced migration in OSCC cells with low endogenous levels of uPAR.ConclusionsThese results show that soluble factors in the tumour microenvironment, such as TGF-β1, PAI-1 and uPA, can influence the ratio of full length and uPAR (II-III) and thereby potentially effect cell migration and invasion. Resolving how uPAR cleavage is controlled is therefore vital for understanding how OSCC progresses and potentially provides new targets for therapy.

Anti-human platelet antigen (HPA)-1a antibodies may affect trophoblast functions crucial for placental development: a laboratory study using an in vitro model

BackgroundFetal and neonatal alloimmune thrombocytopenia (FNAIT) is a bleeding disorder caused by maternal antibodies against paternal human platelet antigens (HPAs) on fetal platelets. Antibodies against HPA-1a are accountable for the majority of FNAIT cases. We have previously shown that high levels of maternal anti-HPA-1a antibodies are associated with clinically significant reduced birth weight in newborn boys. Chronic inflammatory placental lesions are associated with increased risk of reduced birth weight and have previously been reported in connection with FNAIT pregnancies. The HPA-1a epitope is located on integrin β3 that is associated with integrin αIIb (the fibrinogen receptor) on platelets and megakaryocytes. Integrin β3 is also associated with integrin αV forming the αVβ3 integrin heterodimer, the vitronectin receptor, which is expressed on various cell types, including trophoblast cells. It is therefore thinkable that maternal anti-HPA-1a antibodies present during early pregnancy may affect placenta function through binding to the HPA-1a antigen epitope on invasive throphoblasts. The aim of the study was to examine whether interaction of a human anti-HPA-1a monoclonal antibody (mAb) with HPA-1a on trophoblast cells affect adhesion, migration and invasion of extravillous trophoblast cells.MethodsAn in vitro model with human anti-HPA-1a mAb, clone 26.4, and the first trimester extravillous trophoblast cell line HTR8/SVneo was employed. The xCELLigence system was utilized to assess the possible effect of anti-HPA-1a mAb on adhesion and migration of HTR8/SVneo cells. Specially designed chambers precoated with Matrigel were used to assess the effect on the invasive capacity of cells.ResultsWe found that human anti-HPA-1a mAb 26.4 partially inhibits adhesion and migratory capacity of HTR8/SVneo cells.ConclusionsOur findings suggest that anti-HPA-1a antibodies may affect trophoblast functions crucial for normal placental development. Future studies including primary throphoblast cells and polyclonal anti-HPA-1a antibodies are needed to confirm these results.

Human articular chondrocytes express functional leukotriene B4 receptors.

Leukotriene B4 (LTB4) is a potent chemoattractant associated with the development of osteoarthritis (OA), while its receptors BLT1 and BLT2 have been found in synovium and subchondral bone. In this study, we have investigated whether these receptors are also expressed by human cartilage cells and their potential effects on cartilage cells. The expression of LTB4 receptors in native tissue and cultured cells was assessed by immunohistochemistry, immunocytochemistry, polymerase chain reaction (PCR) and electron microscopy. The functional significance of the LTB4 receptor expression was studied by Western blotting, using phospho‐specific antibodies in the presence or absence of receptor antagonists. In further studies, the secretion of pro‐inflammatory cytokines, growth factors and metalloproteinases by LTB4‐stimulated chondrocytes was measured by multiplex protein assays. The effects of LTB4 in cartilage signature gene expression in cultured cells were assessed by quantitative PCR, whereas the LTB4‐promoted matrix synthesis was determined using 3D pellet cultures. Both receptors were present in cultured chondrocytes, as was confirmed by immunolabelling and PCR. The relative quantification by PCR demonstrated a higher expression of the receptors in cells from healthy joints compared with OA cases. The stimulation of cultured chondrocytes with LTB4 resulted in a phosphorylation of downstream transcription factor Erk 1/2, which was reduced after blocking BLT1 signalling. No alteration in the secretion of cytokine and metalloproteinases was recorded after challenging cultured cells with LTB4; likewise, cartilage matrix gene expression and 3D tissue synthesis were unaffected. Chondrocytes express BLT1 and BLT2 receptors, and LTB4 activates the downstream Erk 1/2 pathway by engaging the high‐affinity receptor BLT1. However, any putative role in cartilage biology could not be revealed, and remains to be clarified.

Tissue analyses reveal a potential immune-adjuvant function of FAP-1 positive fibroblasts in non-small cell lung cancer

Objectives Selective targeting of cancer-associated fibroblasts (CAFs) has been proposed to synergize with immune-checkpoint inhibitors. While the roles of CAFs in cancer development are well described, their immune-regulatory properties remain incompletely understood. This study investigates correlations between CAF and immune-markers in tumor stroma from non-small cell lung cancer (NSCLC) patients, and examines whether a combination of CAF and immune cell scores impact patient prognosis. Methods Tumor specimens from 536 primary operable stage I-III NSCLC patients were organized in tissue microarrays. Expression of protein-markers was evaluated by immunohistochemistry. Results Fibroblast and stromal markers PDGFRα, PDGFRβ, FAP-1 and vimentin showed weak correlations while αSMA, and Masson’s trichrome did not correlate with any of the investigated markers. Hierarchical clustering indicated the existence of different CAF-subsets. No relevant correlations were found between any CAF-marker and the immune-markers CD3, CD4, CD8, CD20, CD68, CD1a, CD56, FoxP3 and CD45RO. High density of fibroblast-activation protein positive mesenchymal cells (CAFFAP) was associated with better prognosis in tumors with high infiltration of CD8 and CD3 T-lymphocytes. Conclusions The presented data suggest that CAFs, irrespective of identity, have low influence on the degree of tumor infiltration by inflammatory- and/or immune-cells. However, CAFFAP may exert immuno-adjuvant roles in NSCLC, and targeting CAFs should be cautiously considered.

Radiation-Induced Transformation of Immunoregulatory Networks in the Tumor Stroma

The implementation of novel cancer immunotherapies in the form of immune checkpoint blockers represents a major advancement in the treatment of cancer, and has renewed enthusiasm for identifying new ways to induce antitumor immune responses in patients. Despite the proven efficacy of neutralizing antibodies that target immune checkpoints in some refractory cancers, many patients do not experience therapeutic benefit, possibly owing to a lack of antitumor immune recognition, or to the presence of dominant immunosuppressive mechanisms in the tumor microenvironment (TME). Recent developments in this field have revealed that local radiotherapy (RT) can transform tumors into in situ vaccines, and may help to overcome some of the barriers to tumor-specific immune rejection. RT has the potential to ignite tumor immune recognition by generating immunogenic signals and releasing neoantigens, but the multiple immunosuppressive forces in the TME continue to represent important barriers to successful tumor rejection. In this article, we review the radiation-induced changes in the stromal compartments of tumors that could have an impact on tumor immune attack. Since different RT regimens are known to mediate strikingly different effects on the multifarious elements of the tumor stroma, special emphasis is given to different RT schedules, and the time after treatment at which the effects are measured. A better understanding of TME remodeling following specific RT regimens and the window of opportunity offered by RT will enable optimization of the design of novel treatment combinations.

Abstract 5209: Ionizing radiation abrogates pro-tumorigenic effects from cancer-associated fibroblasts in xenografts

Non-malignant elements of tumors play fundamental roles in both cancer sustainability and responses to therapies. In the context of clinical radiotherapy (RT), all cells that reside in the tumor mass receive radiation, but the impact of cancer-associated fibroblasts (CAFs) to treatment outcomes remains largely unexplored. In this study, we have investigated the effects of radiation on the natural pro-tumorigenic actions of CAFs in animals. Ionizing radiation (IR) was delivered to CAFs either as a single-high dose or fractionated medium doses. Tumor development was compared in xenografts after co-implantation of A549 lung tumor cells with irradiated or non-irradiated human lung CAFs. Quantitative histology and immunohistochemistry was applied to investigate potential biological mechanisms behind tumor growth regulations. As expected, CAFs showed resistance to radiation-induced cell death, however, the enhanced tumorigenic effects observed with admixed non-irradiated CAFs was nullified. Quantitative determinations of parameters such as desmoplasia, angiogenesis, inflammation, innate immunity or tumor cell proliferation revealed enhanced blood vessel density in tumors established with irradiated CAFs. Experiments to ascertain fate of implanted CAFs showed that both irradiated and non-irradiated CAFs only reside at the implantation site during early stages, suggesting that the regulatory functions of admixed CAFs may take place during the initial phases of tumor formation. In this study, we show that CAFs receiving ionizing radiation lose their pro-tumorigenic potential in vivo, affecting angiogenesis and possibly other mechanisms related to tumor engraftment. This finding represent a previously unknown advantageous effect induced by radiotherapy, which adds to the well-known direct cytotoxic effects on transformed epithelial cells. Note: This abstract was not presented at the meeting. Citation Format: Turid Hellevik, Maria T. Grinde, Jorg Vik, Ketil A. Camilio, Inigo Martinez-Zubiaurre. Ionizing radiation abrogates pro-tumorigenic effects from cancer-associated fibroblasts in xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5209. doi:10.1158/1538-7445.AM2017-5209