Sage Journals: Discover world-class research

Abstract

The tumor microenvironment can act so as to stimulate or reject tumor cells. Among the determining factors are cytokines produced, for example, by infiltrating immune cells, tumor cells, and fibroblasts. External radiotherapy has been shown to be able to activate an immune response against tumor cells with cytokine signaling as an important part of the activation. The aim of this study was to evaluate the cytokines present in the tumor microenvironment and whether the cytokine profile changed during tumor regression induced by radioimmunotherapy with the beta emitter ¹⁷⁷Lu. Immunocompetent rats with colon carcinoma tumors were injected with 400 MBq/kg ¹⁷⁷Lu-mAb, and the tumors were excised after 1, 2, 3, 4, 6, or 8 days post injection (4 rats/day on days 1–6 and 8 rats on day 8). Tumors from 10 untreated rats were used as control tissue. The tumors were divided into half: one half was prepared for cytokine analysis with a cytokine array kit and the other half was used for histological analysis. A total of 18 of the 29 cytokines evaluated were detected in this tumor model, and the majority of these act in a pro-inflammatory manner or stimulate the infiltration of immune cells. The differences between treated tumors and control tumors were small, thus the cytokine profile in the untreated tumors did not transfer to an anti-inflammatory profile during tumor regression induced by radioimmunotherapy with ¹⁷⁷Lu. Histological evaluation demonstrated a heterogeneous pattern of ongoing cell death and the formation of granulation tissue.

Keywords

Tumor microenvironment cytokines radioimmunotherapy Lu-177 immunocompetent rat tumor model colon carcinoma

Introduction

Tumors are complex structures consisting of cellular and non-cellular components such as tumor cells (different clones), the extracellular matrix, and stromal cells.^1–3 The tumor microenvironment promotes tumor growth by the secretion of paracrine stimulatory factors, angiogenesis, and immune-mediated interactions.² Cytokines are small signaling proteins that can be produced by a broad range of cells. They can act in either a tumor-promoting or a tumor-rejecting manner and sometimes have dual roles depending on concentration and the combination of cytokines present.⁴ Radiation has been shown to affect cytokine expression,^4–6 thus radiotherapy could change the tumor microenvironment. This change could be part of the immune activation thought to explain the abscopal effect, in which local irradiation results in the regression of a metastasis at a distance from the irradiated site.⁷

Targeted therapy is a cornerstone in personalized medicine with the aim to enhance the specificity of the treatment in order to spare normal tissue. In radioimmunotherapy (RIT), a tumor-targeting antibody is labeled with a radionuclide in order to achieve local irradiation of tumor cells. The effects depend on several factors including target antigen expression and the properties of the radionuclide. In this study, a monoclonal antibody (mAb) was labeled with the beta-emitting radionuclide lutetium-177 (¹⁷⁷Lu) and evaluated in a syngeneic rat colon carcinoma model. This treatment has previously resulted in complete response (CR, defined as non-palpable tumor for at least 1 week) in 17 out of 19 rats.⁸ Analysis of tumors during regression has shown that tumors transformed from dense growth of tumor cells to the formation of granulation tissue with ongoing cell death located mainly in the peripheral parts of the tumor.⁹ The tumors were also infiltrated by neutrophils and lymphocytes.⁹ Analysis of immune cell markers demonstrated that T cells and macrophages augmenting tumor rejection were present to a higher extent than those associated with immune tolerance, and that the number of immune cells decreased as a result of the treatment.¹⁰ After treatment, approximately half of the animals in the syngeneic rat model developed metastases within the follow-up period of 100 days. The number of animals with metastases increased after the depletion of CD8-positive cells,¹¹ indicating that the immune system may be important in preventing the development of metastases. This motivated us to examine the tumor microenvironment prior to and during RIT in the same immunocompetent rat tumor model. The aim of this study was to evaluate the cytokine profile of untreated tumors and of tumors treated with RIT in our immunocompetent rat model to further characterize the effects of ¹⁷⁷Lu-mAb on the immune system.

Methods

The syngeneic rat model

BN7005-H1D2 is a cell line established from a 1,2-dimethylhydrazine-induced colon carcinoma in a Brown Norway (BN) rat.¹² The radiosensitivity of the cell line is similar to that of human colorectal carcinoma cell lines.¹³ We have determined the survival fraction of BN7005-H1D2 cells after 2 Gy irradiation with a ¹³⁷Cs source to be 0.5 (unpublished data). The cells were cultured in RPMI-1640 medium supplemented with 10% fetal calf serum, 1 mM sodium pyruvate, 10 mM HEPES buffer, and 14 mg/L gentamicin (all from Gibco, Invitrogen, Carlsbad, CA) at 37°C, in a humidified environment containing 5% CO₂. The cells were washed in phosphate-buffered saline (PBS) and detached by treatment with trypsin/ethylenediaminetetraacetic acid (EDTA) (PAA Laboratories GmbH, Pasching, Austria).

BN rats are immunocompetent and express the target antigen (Lewis Y) in normal tissues, mainly in the epithelium of the gastrointestinal tract,¹⁴ which is similar to humans.¹⁵ The animals were inoculated with 3 × 10⁵ tumor cells between the abdominal wall and the peritoneum under general anesthesia (isoflurane; Abbott Scandinavia AB, Solna, Sweden). Tumor volumes were calculated as tumor length × tumor width² × 0.4.

The animals were housed under standard conditions and fed with standard pellets and fresh water ad libitum. All experiments were conducted in compliance with European legislation on animal welfare and were approved by the Regional Animal Ethics Committee.

The radioimmunoconjugate

The mouse/human chimeric monoclonal IgG1 antibody BR96 (Seattle Genetics Inc., Seattle, WA) was used. This binds to the tumor-associated antigen Lewis Y that is expressed on the majority of human epithelial tumors. The dissociation constant between BR96 and the cell line used is 4 nM,¹⁶ illustrating the strong binding affinity of the mAb to the tumor cells.

Conjugation of BR96 and S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid (DOTA) was performed according to Forrer et al.¹⁷ Briefly, BR96 was transferred to 0.2 M sodium carbonate buffer, pH 9.5, by repeated centrifugation using an Amicon Ultra-15 filter (molecular weight cut-off 30 kDa; Millipore, Billerica, MA). All empty vials were pretreated with 1% HNO₃, and all buffers were pretreated with Chelex-100 (BioRad, Hercules, CA) to remove metals. The DOTA chelate (2 mg/mL H₂O; Macrocyclics, Dallas, TX) was added to the BR96 antibody (100 mg/mL) at a molar ratio of 3:1 (DOTA:BR96) and incubated for 1 h at 37°C. The conjugate was purified by repeated centrifugation using an Amicon Ultra-15 filter and transferred to 0.25 M ammonium acetate buffer, pH 5.3, and the final concentration was adjusted to 10 mg/mL BR96. Matrix-assisted laser desorption/ionization–mass spectrometry (MALDI-MS) was used to determine the number of DOTA moieties per BR96 molecule, by desalting the sample to 18 MΩ · cm H₂O using a centrifugation filter device, and dividing the increase in molar mass by 688 (the molecular mass of the DOTA chelate).

The immunoreactivity (i.e. the antigen-binding properties) of DOTA-BR96 in relation to BR96 was determined from a saturation binding curve, using BN7005-H1D2 cells as the target antigen. Briefly, increasing concentrations of BR96 and DOTA-BR96 (20 ng/mL–40 µg/mL) were added to the cell plate in triplicate and incubated for 90 min. The bound BR96/DOTA-BR96 conjugate was detected with rabbit anti-human IgG-horseradish peroxidase (HRP) (Dako, Glostrup, Denmark), and the equilibrium binding constant (K_d) was calculated using Prism 5.02 software (GraphPad Software Inc., binding saturation; one site total, non-specific binding and background constrained to a constant value of zero). The immunoreactivity was given by the ratio of the binding constants: K_d(BR96)/K_d(DOTA-BR96).

Both the DOTA-BR96 conjugate and the ¹⁷⁷LuCL₃ solution (MDS Nordion, Vancouver, Canada) were incubated at 45°C for 10 min. The DOTA-BR96 solution was added to the vial containing the radionuclide and incubated at 45°C for 15 min. The reaction was quenched with an excess of DTPA (diethylenetriaminepentaacetic acid) for 5 min. Human serum albumin (HSA; Baxter Medical AB, Kista, Sweden) was added to a final concentration of 1% to prevent radiolysis from affecting the immunoreactivity. High-performance liquid chromatography (HPLC) (7.8 × 300 mm molecular sieving column; Phenomenex SEC S3000, eluted in 50 mM sodium phosphate at 1.0 mL/min) was used to determine the radiochemical purity and to detect signs of aggregation or fragmentation.

RIT with ¹⁷⁷Lu-DOTA-BR96

A total of 38 tumor-bearing male BN rats (Harlan, Horst, Netherlands) were included in the study. Their median weight on the day of administration of RIT was 276 g. In total, 28 rats were treated with 400 MBq/kg body weight of ¹⁷⁷Lu-DOTA-BR96 (150 µg DOTA-BR96 in 0.4 mL saline with 1% HSA) by intravenous injection in the tail vein 13–14 days after cell inoculation. Our previous study showed that 400 MBq/kg body weight resulted in CR in 17 of 19 animals,⁸ and that the maximum tolerable activity was 600 MBq/kg body weight.¹⁸ The treated animals were sacrificed and dissected 1, 2, 3, 4, 6, and 8 days p.i. in groups of 4 animals per day (8 animals on day 8), while 10 untreated animals used as controls were sacrificed and dissected on the day of treatment. The tumors were cut into half, one half was cut into small pieces and placed in vials with protease inhibitor solution (containing 10 µg/mL aprotinin, 10 µg/mL leupeptin, and 10 µg/mL pepstatin from Sigma-Aldrich, St. Louis, MO) and homogenized (DISP 25, from InterMed, Roskilde, Denmark). Triton X-100 (Sigma-Aldrich) was added to the homogenates (final concentration of 1%) before storage at −80°C. The other half was fixed in 4% paraformaldehyde and embedded in paraffin for histological evaluation. In cases where the remaining tumor was small or only scar tissue remained, the whole tumor was used for cytokine analysis.

Cytokine evaluation

The tumor samples with protease inhibitors were centrifuged before determining the protein concentration with a bicinchoninic acid assay. In short, tumor samples were diluted to an expected concentration of 200–1000 µg/mL and added in triplicate to Nunc-Immuno™ Polysorp 96-well plates. Increasing concentrations of protein standard (100–1000 µg/mL, Bovine IgG1; BioRad) were also added in triplicate. A total of 50 parts of bicinchoninic acid solution were mixed with one part copper(II)sulfate pentahydrate solution (both from Sigma-Aldrich) and added to the plates (200 µL/well), which were then incubated for at least 2 h. The plates were read in an enzyme-linked immunosorbent assay (ELISA) spectrophotometer at 562 nm (VERSAmax microplate reader, Molecular Devices, Sunnyvale, CA). The concentrations of the tumor samples were calculated using a linear standard curve obtained from the protein standard.

The Proteome Profiler™ Rat Cytokine Array Kit, Panel A (R&D Systems, Minneapolis, MN) was used according to the manufacturer’s recommendations. Briefly, the cytokine array membrane was blocked with Array Buffer 6 for 1 h on a rocking platform shaker. The tumor sample (400 µg) was added to 0.5-mL Array Buffer 4, and the final volume was adjusted to 1.5 mL with Array Buffer 6. Detection Antibody Cocktail (15 µL) was added to the prepared samples, which were then incubated for 1 h at room temperature. The sample–antibody mixture was added to the membrane, which was incubated at 2°C–8°C on a rocking platform shaker overnight. The membrane was washed three times with wash buffer, for 10 min each. The streptavidin–HRP was diluted in Array Buffer 6 and added to the membrane, which was incubated for 30 min at room temperature on a rocking platform shaker. The membrane was washed three times with Wash Buffer as before. The Chemi Reagent mix was prepared and added to the membrane for 1 min. The Chemi Reagent mix was then removed from the membrane by drying, and the membrane was placed in a plastic sheet protector. The cytokines present were detected and analyzed using a chemiluminescence detector system (FluorChem FC2; Alpha Innotech, San Leandro, CA). The mean luminescence was normalized to reference spots from the same membrane after background correction.

Analysis and evaluation of biomarkers

The paraffin-embedded tumor samples were sectioned (4 µm) and consecutive sections were used for the evaluation of biomarkers. The antibody administered and fragmented DNA were stained as described below. In addition, overall histology was visualized by hematoxylin and eosin staining.

For immunological staining, the sections were incubated at 60°C prior to storage at 4°C. Sections were rehydrated and heat-mediated antigen retrieval was performed using a 2100 Antigen Retriever pressure cooker (Aptum, Southampton, UK) with acidic Target Retrieval Solution (pH 6). The sections were blocked with Peroxidase-Blocking Solution (Dako), the Biotin-Blocking System (Dako), and VECTASTAIN^® goat normal serum (from VECTASTAIN Elite ABC kit (human IgG); Vector Laboratories, Peterborough, UK). The sections were incubated with VECTASTAIN anti-human IgG biotinylated antibody for 30 min at room temperature. After washing, the sections were incubated with VECTASTAIN ABC reagent (prepared 30 min in advance) for 30 min at room temperature. Finally 3,3-diaminobenzidine (DAB; Dako) was added after washing, followed by counterstaining with hematoxylin, dehydration, and mounting.

The fragmented DNA was visualized with the TUNEL assay (TACS^® 2 TdT-Blue Label In Situ Apoptosis Detection Kit, Trevigen, Gaithersburg, MD). The TUNEL assay was performed according to the manufacturer’s recommendations. Briefly, after storage at 4°C, the sections were incubated at 60°C for 5 min, rehydrated, and treated with Proteinase K Solution for 15 min at 37°C. Sections were washed and incubated with Labeling Buffer for 5 min. The sections were washed again and incubated with Labeling Reaction Mix for 1 h at 37°C and washed before incubation with Stop Buffer for 5 min. After washing, the sections were incubated with streptavidin–HRP solution for 10 min at 37°C, and after additional washing, with Blue Label Solution for 3 min. Finally, the sections were counterstained with Nuclear Fast Red for 1 min and dehydrated before mounting.

Results

The radioimmunoconjugate

The immunoreactivity was determined to 0.8 and the average number of DOTA molecules per BR96 antibody was 2.6. The radiochemical purity of ¹⁷⁷Lu-DOTA-BR96 was analyzed using HPLC and found to be 96.2% and there was 3.8% radiolabeled aggregation.

Tumor sampling

All 38 rats developed local tumors between the abdominal wall and peritoneum before the day of treatment (12–13 days after cell inoculation). The mean tumor volume on the day of treatment was 839 mm³ (standard deviation (SD) = 339 mm³). Scar tissue from tumors was collected from rats showing CR on day 4 (one rat), day 6 (one rat), and day 8 (three rats). The rapid treatment response resulted in four additional tumors that could only be used for cytokine analysis (one from day 5 and three from day 8) due to the small size of the tumor.

Cytokine evaluation

A total of 18 of the 29 cytokines evaluated were detected in the tumor samples, listed together with their functions in Table 1. The relative amount of interleukin (IL)-1α, IL-1β, and IL-1ra increased the first days after treatment, while the cytokine-induced neutrophil chemoattractant (CINC)-2 level was elevated between 3 and 6 days p.i., as seen in Figure 1. RANTES initially decreased, with nadir on day 4, before recovering to initial levels. Vascular endothelial growth factor (VEGF), CINC-1, LIX, and macrophage inflammatory protein (MIP)-3α decreased on day 8. CINC-3 and ciliary neurotrophic factor (CNTF) were only detected in a few samples. The relative amounts of the other cytokines detected did not change after treatment with ¹⁷⁷Lu-mAb.

Table 1.

Cytokines present in tumors, cytokine function, and response to ionizing radiation.

Cytokine	Function	Known response to ionizing radiation
CINC-1 (CXCL1, GROα)	Chemokine attracting pro-tumor neutrophils and promoting angiogenesis.^22,23
CINC-2α/β (CXCL3, MIP-2β, GROγ)	Chemokine attracting pro-tumor neutrophils and promoting angiogenesis²³
Fractalkine (CX3CL1)	Chemokine attracting natural killer (NK) cells, dendritic cells (DCs), and T cells,^24–26 reported to have both pro-tumor and anti-tumor effects²⁷
sICAM-1 (CD54)	Inhibits leukocyte adhesion and migration across the endothelium,²⁸ may promote angiogenesis²⁸
IL-1α (IL-1F1)	Pro-inflammatory cytokine,^29,30 promotes infiltration of leucocytes,^30,31 upregulated during hypoxia,³¹ increases tumor invasiveness and angiogenesis³¹	Induced after high-dose radiation^6,29
IL-1β (IL-1F2)	Pro-inflammatory cytokine ^22,30,32 at low doses,³³ agonistic protein to IL-1α,³⁰ increases tumor invasiveness and angiogenesis³¹ at high doses³³	Induced after radiation³²
IL-1ra (IL-1F3)	Antagonist to IL-1α and β^30,33
LIX (CXCL5, ENA-78)	Chemokine attracting pro-tumor neutrophils and promoting angiogenesis²³
L-selectin (CD62L, LECAM-1)	Expressed on T cells and other immune cells and interacts with endothelial cells during leukocyte recruitment³⁴
MIG (CXCL9)	Chemokine promoting recruitment of, for example, cytotoxic T cells and T_H1 cells,^32,35,36 inhibits angiogenesis^35,36	Induced after radiation^32,37
MIP-1α (CCL3)	Chemokine promoting recruitment of, for example, granulocytes, cytotoxic T cells, T helper cells, and NK cells^38–40
MIP-3α (CCL20)	Chemokine promoting recruitment of DCs, B cells, and T cells,^41–43 linked to tumor cell migration by upregulation of MMP-9⁴¹	Induced by high-dose radiation⁶
RANTES (CCL5)	Chemokine promoting recruitment of, for example, T cells, macrophages, eosinophils, and basophils,^39,44 associated with cancer progression and metastasis ^35,44,45
Thymus chemokine (CXCL7)	Promotes angiogenesis^35,36
TIMP-1	Inhibits matrix metalloproteinases involved in tissue remodeling, cell growth, invasion, and metastasis⁴⁶
VEGF	Chemokine promoting recruitment of DCs, myeloid-derived suppressor cells, and T_reg cells,^47,48 promotes angiogenesis^47,48

CXCL: Chemokine (C-X-C motif) ligand; sICAM: soluble intercellular adhesion molecule-1; IL: interleukin; TIMP: tissue inhibitors of metalloproteinases; VEGF: Vascular endothelial growth factor.

Figure 1.

Change of relative amount of detected cytokines. All values are normalized to reference spots.

The cytokines not detected and their function are presented in Table 2. Neither interferon (IFN)-γ nor tumor necrosis factor (TNF)-α was detected. Only minor differences were seen when comparing remaining tumor and scar tissue from day 8 after treatment. Representative cytokine membranes from untreated tumor tissue, RIT-treated tumors, and scar tissue are shown in Figure 2.

Table 2.

Cytokines not detected in tumors, cytokine function, and response to ionizing radiation.

Cytokine	Function	Known response to ionizing radiation
CINC-3 (CXCL2, GROβ)	Chemokine attracting pro-tumor neutrophils and promoting angiogenesis²³
CNTF	Ciliary neurotrophic factor involved in the nervous system
GM-CSF	Stimulates the production of granulocytes and macrophages⁵⁰
IFN-γ	Proinflammatory,²² activates macrophages,⁴⁹ inhibits angiogenesis⁵¹	Increased after local irradiation, directly correlated with tumor burden⁵²
IL-2	Immunostimulatory,⁵⁰ accumulates NK cells in tumor nodules⁵³
IL-3	Stimulates endothelial cell proliferation, migration and formation of blood vessels⁵⁴
IL-4	Polarizes macrophages toward type 2 macrophages (M2) that facilitate tumor growth, invasion, and angiogenesis,⁴⁷ expressed by T_H2 cells⁴⁹
IL-6	Chemokine, promotes recruitment of neutrophils and T cells,⁴⁹ expressed by T_H2 cells⁴⁹	Induced after radiation²⁹
IL-10	Polarizes macrophages toward type 2 macrophages (M2) that facilitate tumor growth, invasion, and angiogenesis,⁴⁷ expressed by T_H2 cells⁴⁹	Induced after lower dose radiation⁶
IL-13	T_H2 cytokine⁵⁵
IL-17	Produces inflammation and neutrophil recruitment,⁵⁶ cooperates with TNF-α to synergistically enhance inflammatory capacity of innate cells⁵⁶
IP-10 (CXCL10)	Remodels vasculature,⁵⁷ chemokine promoting recruitment of, for example, cytotoxic T cells, and T_H1 cells,³² strongly associated with T_H1 cell–based immune response³⁵	Induced after radiation^32,37
TNF-α (TNFSF1A)	Proinflammatory cytokine,^29,32 IL-1, and TNF-α stimulate their own and each other’s production ³³	Induced after radiation,^29,32,37 Induced after high dose radiation⁶

GM-CSF: granulocyte-macrophage colony-stimulating factor; IFN: interferon; IL: interleukin; TNF: tumor necrosis factor; NK cells: natural killer cells.

Figure 2.

Representative cytokine membranes from an (a) untreated tumor, (b and c) RIT-treated tumors, and (d) scar tissue.

Biomarkers within the tumor

Untreated tumors consisted of dense growth of tumor cells and often areas of necrosis in the central part of the tumor (Figure 3(a)). DNA fragmentation was detected (TUNEL staining) in a few cells distributed randomly over the section and in close proximity to necrosis. After the injection of ¹⁷⁷Lu-BR96, ongoing cell death was seen in the peripheral parts of tumor cell areas 1 day p.i., both as single cells and in small clusters. Cell death was mainly found in the same areas as the mAb BR96 (Figure 3(b)). An increase in granulation tissue was also seen as a result of the treatment. These effects were enhanced 2 days p.i. The granulation tissue increased over time, while DNA fragmentation either remained at the same level or decreased to the background level seen in untreated tumors. The few tumors that were excised 8 days p.i. and were sectioned and stained consisted mainly of granulation tissue and showed ongoing cell death in tumor cell areas (Figure 3(c)).

Figure 3.

Tumor histology and distribution of therapeutic antibody ¹⁷⁷Lu-BR96. (a) Untreated tumor tissue consisted of dense tumor cell growth. (b) Ongoing cell death was seen in areas with targeting antibody 1 day p.i. (c) Remaining tumors consisted mainly of granulation tissue 8 days p.i. Scale bar denotes 50 µm.

Discussion

The tumor immune contexture impacts the prognosis as seen in patients with colorectal cancer, and tumors can be divided into three groups based on this finding: (1) “immunogenic tumors” with anti-tumor immune cells result in a good prognosis, (2) “inflammatory tumors” with production of angiogenic and immunosuppressive molecules result in poor prognosis, and (3) “escaping tumors” with downregulation of antigen-presenting molecules leading to avoidance of an immune attack result in intermediate prognosis.¹⁹ The tumor microenvironment contains a large variety of cells, and cytokines can be secreted by all cells present in the tumors, including cancer-associated fibroblasts.²⁰ Cytokines in the tumor microenvironment plays an important part in stimulating tumor growth and orchestrating the immune contexture.⁴ These can be divided into functional groups: pro-inflammatory (TNF-α, IL-1α/β, and IL-17), anti-inflammatory (IL-4, IL-10, and transforming growth factor (TGF)-β), angiogenic (VEGF, TNF-α, and fibroblast growth factor (FGF)), pro-fibrotic (IL-6 and TGF-β), immune (IL-2, IL-4, and IL-7), and hematopoietic (colony-stimulating factor 1 (CSF1), granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-3, and erythropoietin (EPO)).⁴ Some cytokines have been shown to have dual roles depending on dose and other cytokines present in the tissue,^4,21 adding to the complexity of the cytokine environment and making the assessment of their true function difficult.

Cytokine analysis showed that 18 of the 29 cytokines evaluated were detected in the tumor samples. These were mainly involved in processes of infiltration of immune cells and angiogenesis. The presence of chemokines promoting the recruitment of leukocytes corresponded with earlier studies showing infiltration of neutrophils,⁹ T cells, and macrophages¹⁰ in this tumor model. The cytokine pattern was considered to be neither pro-inflammatory (described as consisting of IL-6, IL-17, IFN-γ, TNF-α, IL-1α/β among others) nor anti-inflammatory (containing cytokines like IL-1ra, IL-14, IL-5, IL-10, IL-11, IL-13).⁴ Simultaneous immune stimulation and suppression has been seen in patients with advanced-stage tumors (consisting of increased levels of macrophage migration inhibitory factor (MIF), TNF-α, IL-6, IL-8, IL-10, IL-18 and TGF-β,⁴⁹ which has been interpreted as an inflammatory environment with infiltrating T_H17 cells that are linked to chronic inflammation and autoimmune disease. However, this pattern does not correspond to the detected cytokines in this study.

The differences in cytokine content between untreated and RIT-treated tumors were small, indicating that the cytokine profile of untreated tumors was not transformed during tumor regression induced by RIT, especially since other cytokines present are thought to be involved in the same processes, that is, chemoattraction of immune cells and angiogenesis. RANTES was the chemokine with the most pronounced decrease after RIT, thus indicating a reduction in infiltration of T cells and macrophages. However, other chemokines with proposed similar functions were unaffected. Thus, it is not possible to draw any detailed conclusions until the roles of cytokines have been further specified. IFN-γ and TNF-α have been shown to promote the immune response of radiotherapy,⁵ and thus the lack of both of these cytokines after RIT implicates that no such immune stimulation was initiated.

The cytokine array was chosen as it enabled simultaneous screening of many cytokines using a small amount of tumor sample; however, it does not provide any information on concentration. Thus, although the analysis showed higher density of IL-1ra than IL-1α and IL-1β, it is not clear whether the levels are high enough to block signaling, especially since abundant levels of IL-1ra are needed to functionally inhibit the signaling.⁵⁸

The histological evaluation demonstrated ongoing cell death in peripheral parts of the tumor, while central parts remained unaffected, as seen previously in another study with the same tumor model.⁹ Staining of the injected mAb in this study followed the same pattern as seen previously when evaluated by autoradiography and could thus be used as a marker for radioactivity when autoradiography cannot be performed. The response to RIT in the tumor microenvironment would most likely follow the same heterogeneous pattern as the activity, implying that local changes in cytokine expression can be at least partly masked due to the homogenization of the tumor samples for cytokine analysis. In addition, at later time points in this study, there is a risk of selection of tumors that do not respond or respond slower to RIT than the tumors that have been rejected.

Radiation has been shown to be able to change cytokine expression, which could lead to remodeling of the stromal and vascular tissue, and changes in the function of the immune cells present in the tumor.⁵⁹ Both the innate and adaptive arms of the immune system can be triggered by radiation, but the optimal radiation regimen to activate the immune response remains to be defined.³² Studies of tumor cell lines indicate that the secretion of cytokines after radiation seems to be cell-line specific.⁶⁰ In the in vivo situation, the tumor contains different tumor cell clones and normal cell types, making it difficult to compare the results of in vitro and in vivo studies. In addition, studies of external beam radiotherapy have shown that different radiation doses induce different cytokine profiles,^4–6 without any linear correlation.⁴ For example, T_H1- and T_H2-related cytokines decreased after a low absorbed dose, but increased after a high absorbed dose.⁶¹ A local high dose (20 Gy) has also been shown to trigger the production of type I IFNs and upregulate CXCL9 and CXCL10,⁵ leading to recruitment of cytotoxic T cells. Low-dose irradiation (<2 Gy) tends to promote anti-inflammatory responses associated with reduced production of NO and IL-1β and an increase in IL-10 and TGF-β.⁵ However, these patterns were not found in our tumor model, in which the expression of CXCL9 did not change after irradiation and neither CXCL10 nor IL-10 was detected (TGF-β was not included in the cytokine array). The expected mean absorbed dose rate in this study is in the range 0.2–0.6 Gy/h at 24-h p.i.⁹ and the total dose >25 Gy (based on unpublished data). However, since dose rates and particle energy differ between external beam radiotherapy and radionuclide therapy such as RIT, it is difficult to make any general comparisons between the two radiation modalities. It might be that the low dose rate cannot trigger an immune response or that the response is delayed due to the slow distribution of the radiolabeled mAb and the low dose rate. In the latter case, the lack of significant differences in cytokine expression between untreated and treated tumors in this study could be due to the short follow-up time. However, a longer follow-up time was not possible due to the rapid tumor regression after RIT in this syngeneic rat model.

Conclusion

The cytokine profile of untreated tumors was not transformed during tumor regression by RIT with ¹⁷⁷Lu in this colon carcinoma model.

Footnotes

Acknowledgements

The authors would like to thank Dr Peter Senter (Seattle Genetics Inc.) for kindly providing the monoclonal antibody BR96.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Ethical approval

All applicable international, national, and institutional guidelines for the care and use of animals were followed. This article does not contain any study with human participants performed by any of the authors.

Funding

This study was funded by the Swedish Cancer Society, Mrs Berta Kamprad’s Foundation, Governmental Founding of Clinical Research within the Swedish National Health Service, King Gustaf V’s Jubilee Foundation, The Lund University Medical Faculty Foundation, and The Lund University Hospital Fund.

References

Dudas

Supportive and rejective functions of tumor stroma on tumor cell growth, survival, and invasivity: the cancer evolution. Front Oncol 2015; 5: 44.

Gerdes

Sood

Sevinsky

. Emerging understanding of multiscale tumor heterogeneity. Front Oncol 2014; 4: 366.

Junttila

De Sauvage

FJ.

Influence of tumour micro-environment heterogeneity on therapeutic response. Nature 2013; 501(7467): 346–354.

Schaue

Kachikwu

McBride

WH.

Cytokines in radiobiological responses: a review. Radiat Res 2012; 178(6): 505–523.

Haikerwal

Hagekyriakou

MacManus

. Building immunity to cancer with radiation therapy. Cancer Lett 2015; 368(2): 198–208.

Rodel

Frey

Multhoff

. Contribution of the immune system to bystander and non-targeted effects of ionizing radiation. Cancer Lett 2015; 356(1): 105–113.

Siva

MacManus

Martin

. Abscopal effects of radiation therapy: a clinical review for the radiobiologist. Cancer Lett 2015; 356(1): 82–90.

Eriksson

Ohlsson

Nilsson

. Treatment with unlabeled mAb BR96 after radioimmunotherapy with 177Lu-DOTA-BR96 in a syngeneic rat colon carcinoma model. Cancer Biother Radiopharm 2012; 27(3): 175–182.

Elgstrom

Ljungberg

Eriksson

. Change in cell death markers during (177)Lu-mAb radioimmunotherapy-induced rejection of syngeneic rat colon carcinoma. Cancer Biother Radiopharm 2014; 29(4): 143–152.

10.

Elgstrom

Eriksson

Ljungberg

. Evaluation of immune cell markers in tumor tissue treated with radioimmunotherapy in an immunocompetent rat colon carcinoma model. EJNMMI Res 2015; 5(1): 47.

11.

Elgstrom

Eriksson

Ohlsson

. Role of CD8-positive cells in radioimmunotherapy utilizing (177)Lu-mAbs in an immunocompetent rat colon carcinoma model. EJNMMI Res 2015; 5: 3.

12.

Brodin

Jansson

Hedlund

. Use of a monoclonal rat anti-mouse Ig light chain (RAMOL-1) antibody reduces background binding in immunohistochemical and fluorescent antibody analysis. J Histochem Cytochem 1989; 37(7): 1013–1024.

13.

Deschavanne

Fertil

A review of human cell radiosensitivity in vitro. Int J Radiat Oncol Biol Phys 1996; 34(1): 251–266.

14.

Sjogren

Isaksson

Willner

. Antitumor activity of carcinoma-reactive BR96-doxorubicin conjugate against human carcinomas in athymic mice and rats and syngeneic rat carcinomas in immunocompetent rats. Cancer Res 1997; 57(20): 4530–4536.

15.

Hellstrom

Garrigues

. Highly tumor-reactive, internalizing, mouse monoclonal antibodies to Le(y)-related cell surface antigens. Cancer Res 1990; 50(7): 2183–2190.

16.

Eriksson

Ohlsson

Nilsson

. Repeated radioimmunotherapy with 177Lu-DOTA-BR96 in a syngeneic rat colon carcinoma model. Cancer Biother Radiopharm 2012; 27(2): 134–140.

17.

Forrer

Chen

Fani

. In vitro characterization of (177)Lu-radiolabelled chimeric anti-CD20 monoclonal antibody and a preliminary dosimetry study. Eur J Nucl Med Mol Imaging 2009; 36(9): 1443–1452.

18.

Martensson

Nilsson

Ohlsson

. High-dose radioimmunotherapy combined with extracorporeal depletion in a syngeneic rat tumor model: evaluation of toxicity, therapeutic effect, and tumor model. Cancer 2010; 116(4 Suppl.): 1043–1052.

19.

Becht

Giraldo

Dieu-Nosjean

. Cancer immune contexture and immunotherapy. Curr Opin Immunol 2016; 39: 7–13.

20.

Smith

Kang

The metastasis-promoting roles of tumor-associated immune cells. J Mol Med 2013; 91(4): 411–429.

21.

Le Bitoux

Stamenkovic

Tumor-host interactions: the role of inflammation. Histochem Cell Biol 2008; 130(6): 1079–1090.

22.

Michalak

Wender

Michalowska-Wender

. Blood-brain barrier breakdown and cerebellar degeneration in the course of experimental neoplastic disease. Are circulating Cytokine-Induced Neutrophil Chemoattractant-1 (CINC-1) and -2alpha(CINC-2alpha) the involved mediators? Folia Neuropathol 2010; 48(2): 93–103.

23.

Verbeke

Geboes

Van Damme

. The role of CXC chemokines in the transition of chronic inflammation to esophageal and gastric cancer. Biochim Biophys Acta 2012; 1825(1): 117–129.

24.

Park

Lee

Yoon

JH.

High expression of CX3CL1 by tumor cells correlates with a good prognosis and increased tumor-infiltrating CD8+ T cells, natural killer cells, and dendritic cells in breast carcinoma. J Surg Oncol 2012; 106(4): 386–392.

25.

Deng

Chen

. CXCR6/CXCL16 functions as a regulator in metastasis and progression of cancer. Biochim Biophys Acta 2010; 1806(1): 42–49.

26.

Hyakudomi

Matsubara

Hyakudomi

. Increased expression of fractalkine is correlated with a better prognosis and an increased number of both CD8+ T cells and natural killer cells in gastric adenocarcinoma. Ann Surg Oncol 2008; 15(6): 1775–1782.

27.

Tsang

Chan

. CX3CL1 expression is associated with poor outcome in breast cancer patients. Breast Cancer Res Treat 2013; 140(3): 495–504.

28.

Witkowska

Borawska

MH.

Soluble intercellular adhesion molecule-1 (sICAM-1): an overview. Eur Cytokine Netw 2004; 15(2): 91–98.

29.

Burnette

Liang

Lee

. The efficacy of radiotherapy relies upon induction of type I interferon-dependent innate and adaptive immunity. Cancer Res 2011; 71(7): 2488–2496.

30.

Voronov

Carmi

Apte

RN.

The role IL-1 in tumor-mediated angiogenesis. Front Physiol 2014; 5: 114.

31.

Rider

Carmi

Voronov

. Interleukin-1alpha. Semin Immunol 2013; 25(6): 430–438.

32.

Formenti

Demaria

Combining radiotherapy and cancer immunotherapy: a paradigm shift. J Natl Cancer Inst 2013; 105(4): 256–265.

33.

Apte

Voronov

Is interleukin-1 a good or bad “guy” in tumor immunobiology and immunotherapy?

Immunol Rev 2008; 222: 222–241.

34.

Paschos

Canovas

Bird

NC.

The engagement of selectins and their ligands in colorectal cancer liver metastases. J Cell Mol Med 2010; 14(1–2): 165–174.

35.

Chow

Luster

AD.

Chemokines in cancer. Cancer Immunol Res 2014; 2(12): 1125–1131.

36.

Rainczuk

Rao

Gathercole

. The emerging role of CXC chemokines in epithelial ovarian cancer. Reproduction 2012; 144(3): 303–317.

37.

Park

Yee

Lee

KM.

The effect of radiation on the immune response to cancers. Int J Mol Sci 2014; 15(1): 927–943.

38.

Borsig

Wolf

Roblek

. Inflammatory chemokines and metastasis–tracing the accessory. Oncogene 2014; 33(25): 3217–3224.

39.

Draghiciu

Walczak

Hoogeboom

. Therapeutic immunization and local low-dose tumor irradiation, a reinforcing combination. Int J Cancer 2014; 134(4): 859–872.

40.

Terpos

Politou

Viniou

. Significance of macrophage inflammatory protein-1 alpha (MIP-1alpha) in multiple myeloma. Leuk Lymphoma 2005; 46(12): 1699–1707.

41.

Campbell

Albo

Kimsey

. Macrophage inflammatory protein-3alpha promotes pancreatic cancer cell invasion. J Surg Res 2005; 123(1): 96–101.

42.

Arab

Mojarrad

Motamedi

. Tumour regression induced by co-administration of MIP-3α and CpG in an experimental model of colon carcinoma. Scand J Immunol 2013; 78(1): 28–34.

43.

Schutyser

Struyf

Van Damme

The CC chemokine CCL20 and its receptor CCR6. Cytokine Growth Factor Rev 2003; 14(5): 409–426.

44.

Aldinucci

Colombatti

The inflammatory chemokine CCL5 and cancer progression. Mediators Inflamm 2014; 2014: 292376.

45.

Laubli

Spanaus

Borsig

Selectin-mediated activation of endothelial cells induces expression of CCL5 and promotes metastasis through recruitment of monocytes. Blood 2009; 114(20): 4583–4591.

46.

Ries

Cytokine functions of TIMP-1. Cell Mol Life Sci 2014; 71(4): 659–672.

47.

Terme

Colussi

Marcheteau

. Modulation of immunity by antiangiogenic molecules in cancer. Clin Dev Immunol 2012; 2012: 492920.

48.

Voron

Marcheteau

Pernot

. Control of the immune response by pro-angiogenic factors. Front Oncol 2014; 4: 70.

49.

Lippitz

BE.

Cytokine patterns in patients with cancer: a systematic review. Lancet Oncol 2013; 14(6): e218–e228.

50.

Vacchelli

Aranda

Obrist

. Trial watch: immunostimulatory cytokines in cancer therapy. Oncoimmunology 2014; 3: e29030.

51.

Ngiow

Teng

Smyth

MJ.

A balance of interleukin-12 and -23 in cancer. Trends Immunol 2013; 34(11): 548–555.

52.

Gerber

Sedlacek

Cron

. IFN-gamma mediates the antitumor effects of radiation therapy in a murine colon tumor. Am J Pathol 2013; 182(6): 2345–2354.

53.

Maghazachi

AA.

Role of chemokines in the biology of natural killer cells. Curr Top Microbiol Immunol 2010; 341: 37–58.

54.

Broughton

Dhagat

Hercus

. The GM-CSF/IL-3/IL-5 cytokine receptor family: from ligand recognition to initiation of signaling. Immunol Rev 2012; 250(1): 277–302.

55.

Terabe

Park

Berzofsky

JA.

Role of IL-13 in regulation of anti-tumor immunity and tumor growth. Cancer Immunol Immunother 2004; 53(2): 79–85.

56.

Reynolds

Angkasekwinai

Dong

IL-17 family member cytokines: regulation and function in innate immunity. Cytokine Growth Factor Rev 2010; 21(6): 413–423.

57.

Kershaw

Devaud

John

. Enhancing immunotherapy using chemotherapy and radiation to modify the tumor microenvironment. Oncoimmunology 2013; 2(9): e25962.

58.

Arend

, The balance between IL-1 and IL-1Ra in disease. Cytokine Growth Factor Rev 2002; 13(4–5): 323–340.

59.

Ahmed

Hodge

Guha

. Harnessing the potential of radiation-induced immune modulation for cancer therapy. Cancer Immunol Res 2013; 1(5): 280–284.

60.

Desai

Kumar

Laskar

. Cytokine profile of conditioned medium from human tumor cell lines after acute and fractionated doses of gamma radiation and its effect on survival of bystander tumor cells. Cytokine 2013; 61(1): 54–62.

61.

Bogdandi

Balogh

Felgyinszki

. Effects of low-dose radiation on the immune system of mice after total-body irradiation. Radiat Res 2010; 174(4): 480–489.

Cytokine evaluation in untreated and radioimmunotherapy-treated tumors in an immunocompetent rat model

Abstract

Keywords

Introduction

Methods

The syngeneic rat model

The radioimmunoconjugate

RIT with 177Lu-DOTA-BR96

Cytokine evaluation

Analysis and evaluation of biomarkers

Results

The radioimmunoconjugate

Tumor sampling

Cytokine evaluation

Biomarkers within the tumor

Discussion

Conclusion

Footnotes

Acknowledgements

Declaration of conflicting interests

Ethical approval

Funding

References

RIT with ¹⁷⁷Lu-DOTA-BR96