Spider venom administration impairs glioblastoma growth and modulates immune response in a non-clinical model

Molecules from animal venoms are promising candidates for the development of new drugs. Previous in vitro studies have shown that the venom of the spider Phoneutria nigriventer (PnV) is a potential source of antineoplastic components with activity in glioblastoma (GB) cell lines. In the present work, the effects of PnV on tumor development were established in vivo using a xenogeneic model. Human GB (NG97, the most responsive line in the previous study) cells were inoculated (s.c.) on the back of RAG−/− mice. PnV (100 µg/Kg) was administrated every 48 h (i.p.) for 14 days and several endpoints were evaluated: tumor growth and metabolism (by microPET/CT, using 18F-FDG), tumor weight and volume, histopathology, blood analysis, percentage and profile of macrophages, neutrophils and NK cells isolated from the spleen (by flow cytometry) and the presence of macrophages (Iba-1 positive) within/surrounding the tumor. The effect of venom was also evaluated on macrophages in vitro. Tumors from PnV-treated animals were smaller and did not uptake detectable amounts of 18F-FDG, compared to control (untreated). PnV-tumor was necrotic, lacking the histopathological characteristics typical of GB. Since in classic chemotherapies it is observed a decrease in immune response, methotrexate (MTX) was used only to compare the PnV effects on innate immune cells with a highly immunosuppressive antineoplastic drug. The venom increased monocytes, neutrophils and NK cells, and this effect was the opposite of that observed in the animals treated with MTX. PnV increased the number of macrophages in the tumor, while did not increase in the spleen, suggesting that PnV-activated macrophages were led preferentially to the tumor. Macrophages were activated in vitro by the venom, becoming more phagocytic; these results confirm that this cell is a target of PnV components. Spleen and in vitro PnV-activated macrophages were different of M1, since they did not produce pro- and anti-inflammatory cytokines. Studies in progress are selecting the venom molecules with antitumor and immunomodulatory effects and trying to better understand their mechanisms. The identification, optimization and synthesis of antineoplastic drugs from PnV molecules may lead to a new multitarget chemotherapy. Glioblastoma is associated with high morbidity and mortality; therefore, research to develop new treatments has great social relevance. Natural products and their derivatives represent over one-third of all new molecular entities approved by FDA. However, arthropod venoms are underexploited, although they are a rich source of new molecules. A recent in vitro screening of the Phoneutria nigriventer spider venom (PnV) antitumor effects by our group has shown that the venom significantly affected glioblastoma cell lines. Therefore, it would be relevant to establish the effects of PnV on tumor development in vivo, considering the complex neoplastic microenvironment. The venom was effective at impairing tumor development in murine xenogeneic model, activating the innate immune response and increasing tumor infiltrating macrophages. In addition, PnV activated macrophages in vitro for a different profile of M1. These activated PnV-macrophages have potential to fight the tumor without promoting tumorigenesis. Studies in progress are selecting the venom molecules with antitumor and immunomodulatory effects and trying to better understand their mechanisms. We aim to synthesize and carry out a formulation with these antineoplastic molecules for clinical trials. Spider venom biomolecules induced smaller and necrotic xenogeneic GB; spider venom activated the innate immune system; venom increased blood monocytes and the migration of macrophages to the tumor; activated PnV-macrophages have a profile different of M1 and have a potential to fight the tumor without promote tumorigenesis.

CT scan (AlbiraSi, Bruker) was performed on the day 21 (at the end of the treatments), at the Preclinical Image Laboratory (LIP) -Medical Sciences School, UNICAMP. The equipment has three attached image modes: PET, SPECT and CT. For this research, the PET and CT modalities were used. Prior to administration of the radiotracer 18 F-FDG (fluorodeoxyglucose), mice were fasted for at least 6.0 h. The 18 F-FDG was injected into the intraorbital plexus (i.v.) 40 min before the scan start, for the biodistribution of the radiotracer. The animal was placed in a dedicated isoflurane administration system (MIP/Anesthesia Technologies) for inhalation anesthesia. With no further motor stimuli, the radiotracer was administered and throughout the experiment the animal was maintained under isoflurane and oxygen administration. In addition, the temperature of the animal's environment was constantly checked, reaching about 23 °C (73.4 °F). The protocols chosen (selected) to acquire the images of the animals were: PET in single mode of 20 minutes and CT of 35 kV, 400 uA and 1024 projections. The images were reconstructed in the AlbiraSi system reconstruction mode. For CT, two images were generated: CT Low (for attenuation correction) and CT High (for corregistration and fusion). For PET, the MLEM (Maximum Likelihood Estimation Method), with voxel size 20 × 20 × 0.5, 12 iterations and with all the corrections activated (scatter correction, decay correction and attenuation correction with CT Low), was used. Finally, for the quantification of the images, the PET fusion image with attenuation correction and CT High were used. To quantify the uptake of the radiotracer present in the volume, the SUV calculation (standardized uptake value) is adopted. This metric can be defined as the ratio between the concentration of activity present in the structure (Bq/volume) and the activity injected (Bq) in the individual multiplied by its respective mass (grams for animals and kilograms for humans). In the present work, the SUV average was quantified, that is, the value of the average concentration present in the structure of interest.
tumor histopathology. After PET-CT, the animals were anesthetized with ketamine (80 mg/Kg) and xylazine (10 mg/Kg) and sacrificed. The tumors were dissected and fixed in phosphate-buffered 10% formalin. Samples were then dehydrated by graded ethanol and embedded in Paraplast Plus. Sections of 5 µm were examined after staining with hematoxylin and eosin (H&E).
Immunofluorescence: detection of tumor associated macrophages. The level of Iba-1 (a macrophage marker) was investigated in 5 μm paraffin sections. Sections were deparaffinized and boiled in citrate buffer (1 mM; pH 6.0) for 30 min in a steam cooker for antigenic recovery. Rabbit polyclonal antibody against Iba-1 (1:100 dilution, FUJIFILM Wako Chemicals U.S.A Corp, Richmond, VA, EUA, #019-19741) was used as primary antibody (negative controls were maintained in PBS), followed by secondary anti-rabbit Cy5 conjugated (Jackson Immuno Research, #711-175-152). Images were acquired using a microscope (Leica DM5500B) coupled to a camera (DFC345 FX) and Image-ProPlus 6.0 software (Media Cybemetics, USA). cell culture and treatments. Macrophages were obtained from differentiation of murine bone marrow cells cultivated with L929 cells supernatant, according to Marim et al. 20 . Bone marrow precursors were collected from femurs of 7-10 weeks old male C57BL/6 J mice. After collection, the marrow cell suspension was cultured in Petri dishes with 10 mL of Iscove's Modified Dulbecco's Media (IMDM) supplemented with 20% fetal bovine serum (FBS) and 30% L929 cell supernatant (as a source of M-CSF). After four days, another 10 mL of fresh medium was added in the same proportions. This culture was incubated for seven days at 37 °C and 5% of CO 2 and thereafter these cells were washed with PBS and adherent cells (differentiated macrophages) were harvested with a cell scraper and transferred to a 24 wells plate (2 × 10 5 cells per well) with a round cover slip at the bottom. phagocytosis assay. For this test, the pHrodo Red E. coli BioParticles Conjugate for Phagocytosis assay was used (Thermo Fisher Scientific, #P35361). The macrophages were pre-activated with 20 ng/mL recombinant mouse IFN-γ (BD #554587) for 48 h and then received the following treatments: PBS only (M0 profile control), E. coli O26:B6 LPS 1 µg/mL (used as a canonical positive control of macrophages activation to M1 profile 21 to compare with PnV effects), or PnV 14 µg/mL. After 24 h, treatments were removed, macrophages were washed with PBS and incubated for 4 h with 50 µg bioparticles. The cells were then extensively washed, fixed with paraformaldehyde for 15 min and labeled with Iba-1 by immunofluorescence. Briefly, cells were incubated with blocking buffer (PBS with 1% BSA and 0.3% Triton X100) for 2 h, followed by anti-Iba-1 (Wako #019-19741; 1:700) (2020) 10:5876 | https://doi.org/10.1038/s41598-020-62620-9 www.nature.com/scientificreports www.nature.com/scientificreports/ overnight, and secondary antibody (Cy2-Goat anti-rabbit, Jackson ImmunoResearch, #111225144; 1:1000) for 1 h. Nuclei were stained with DAPI (1:1000) for 5 min. Macrophages were washed and sliders were mounted using a mounting medium for fluorescence. Images were acquired in the Cytation 5 -Cell Imaging Multi-Mode reader (BioTek, Winooski, VT, USA). For quantification, images of three randomly selected fields per treatment were taken and integrate analysis of pixels was determined using Image J software (version 1.52p, NIH, USA).

ELISA (enzyme-linked immunosorbent assay).
The quantification of TNF-alpha and IL-10 in the culture medium was performed using R&D Systems -DuoSet immunoassay kits in accordance with the manufacturer's instructions. Monoclonal antibodies specific for mouse TNF-alpha and IL-10 were pre-coated on the microplate. Standards, controls and samples were pipetted into the wells and any TNF-alpha or IL-10 present was bound to the immobilized antibody. Following washing, a substrate solution was added. The enzyme reaction yields a blue product that turns yellow when the stop solution is added. The intensity of the color measured is in proportion to the amount of mouse cytokines bound at the initial steps. The sample values were then read off the standard curve in a Multiskan Go microplate spectrophotometer (Thermo Fisher Scientific, Inc., Waltham, MA, USA). All dosages were performed in triplicate.

Venom purification and MTT (cell viability assay).
The initial fractionation of the crude venom was performed by the Amicon Ultra Centrifugal Filter (#UFC801008; Thermo Fisher Scientific, Suwannee, GA). This procedure consisted of separating crude venom by molecular mass using molecular filters, generating three main fractions named: F1 (low weight, less than 3 kDa), F2 (intermediate weight, between 3 and 10 kDa) and F3 (high weight, more than 10 kDa). From these, cell viability assay (MTT; described below) was conducted to select the most significant fraction, considering the antineoplastic effects; Then, F1 and F2 were chosen and F3 was eliminated. Further purification of F1 and F2 was carried out: reversed phase HPLC was performed using a Shimadzu VP-ODS column, 0.1% trifluoroacetic acid (TFA) as mobile phase and 90% acetonitrile 0.1% TFA as eluent. More purified components were obtained, named subfractions 1-12 (SF1-SF12).
Thiazolyl Blue Tetrazolium Bromide (MTT) was used to determine if Fs and SFs would alter cell viability. NG97 cells were seeded in 96-well plates at an initial density of 5 × 10 5 cells per well and incubated for 72 h at 37 °C for confluence (in IMDM supplemented with 10% FBS and 100 UI/ml penicillin and streptomycin). Then, cells were treated with PnV (14 µg/ml), F1, F2, F3 or all twelve subfractions (SF1-SF12; 0.1 µg/ml) for 5 h, while control cells were maintained in medium. After removal of the treatments, MTT was added to each well and incubated at 37 °C for 4 h according to the manufacturer's protocol. Thereafter, acidified isopropanol was added to each well to solubilize the blue formazan crystals. Absorbance at 540 nm was determined on a Multiskan GO spectrophotometer.
Statistic. Values were analyzed using the GraphPad Prism software package (San Diego, CA, USA; v. 5.0).
The level of significance was obtained by one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparisons test. Error bars show the standard error of the mean (SEM). Unpaired Student's t-test was used to compare each treatment with the control. A p-value <0.05 indicated statistical significance.

Results
Quality and reproducibility of venom. HPLC demonstrated that there was no significant difference between the two extracted venom samples. The samples were the same used in the experiments published before by Santos et al. [14], however the quality of PnV lots was established using 214 nm wavelength (Supplementary Fig. 1).
PnV administration increased body weight gain and improved survival, compared to control. The control group recorded gradual loss of weight (data were not significant) after inoculation of the cells (day 1) until the end of the experiment (day 20, last recorded weight). On day 7, the treatments were started. The PnV group presented weight loss on day 7 in relation to day 1, followed by progressive recovery after the beginning of the treatment, being significant when comparing day 20 with day 7 (p < 0,05) (Fig. 1A). Control animals showed 40% of mortality (due to general clinical condition caused by the tumor), while PnV group had no deaths (Fig. 1B). Figure 1(C-G) shows representative animals (schematically and per photograph) of the control (C and D) and PnV (E -G), at the end of the experiment (day 21). PET-CT images showed that control animals developed large tumors ( Fig. 2A,B,D), with intense uptake of 18 F-FDG (Fig. 2C,E), suggesting a high metabolism, expected for these tumor cells. Mice receiving PnV therapy showed smaller tumors (Fig. 2F,G,L), which were negative for 18 F-FDG (Fig. 2H,J), indicating slower metabolism compared to control. Figure 2A,B represent 3D reconstructions of CT for each group. The graphs K and L of Fig. 2 show the statistic results of tumor weight and volume, respectively, and graph M shows the results of 18 F-FDG uptake (SUV average). Representative animals of control and PnV groups are also shown by 3D animation. Table 1 shows the weight and volume of individual tumors.

Mice treated with pnV developed smaller and 18 F-FDG-negative tumors, compared to control and MTX.
Supplementary Figure 2 shows a PnV-treated animal that did not develop tumor in the back (the site of cells inoculation), however, interestingly, only this mouse developed a brain tumor, as shown by CT (B -D) and PET-18 F-FDG (E -G) (panel A represents a 3D reconstruction). chemotherapy with pnV induced tumor necrosis. Histopathological analysis has showed that control animals developed tumors with typical GB characteristics ( Fig. 3A-C). Panel A of Fig. 3 shows a hypercellular tumor composed of a variety of cell types (cell polymorphism) (asterisks), including small cells with www.nature.com/scientificreports www.nature.com/scientificreports/ hyperchromatic nuclei, cells with pink glial-like cytoplasm, and endothelial cells. In panel B, it is possible to see an area with a pink cellular necrosis at the center (asterisk) with tumor cell palisading (arrow); also, glomeruloid vascular proliferation of endothelial cells is present (arrow on panel C). www.nature.com/scientificreports www.nature.com/scientificreports/ Tumors of animals treated with PnV have lost most of the typical characteristics of glioblastoma; It has showed many congestive blood vessels (arrow in panels D and E of Fig. 3) and large areas of necrosis (asterisks in panels E and F).
Treatment with PnV altered blood cell count, leading to a different profile compared to MTX. In general, chemotherapy with PnV induced an increase in blood cells, whereas MTX induced a decrease (expected for traditional chemotherapy) (Fig. 4); The treatments did not significantly alter the total leucocytes, however it can be observed that, qualitatively, PnV-treated animals were prone to increase them, while MTX showed a decrease (Fig. 4A). The same profile was observed for the number of neutrophils (Fig. 4B). The percentage of cells, on the other hand, showed that PnV did not induce change, whereas MTX induced a significant neutropenia (Fig. 4C). Treatment with PnV induced a significant increase in the number of monocytes and percentage of cells (compared to control and MTX) (Fig. 4D,E). For eosinophils, no significant alterations were observed (Fig. 4F,G).
Compared to the control, PnV did not change the number of erythrocytes. However, the number of red cells was significantly lower in the animals receiving MTX (Fig. 5A). A similar result was observed for hemoglobin and hematocrit (Fig. 5B,C). PnV did not alter the platelets, comparing to the control animals. MTX, on the other hand, induced a significant increase in platelets compared to all other groups (Fig. 5D).
Chemotherapy with PnV induced alterations in hepatic, but not in kidney, functional parameters. Hepatic function markers, GOT and GTP, were also measured in serum (Fig. 5E,F). PnV induced a significant increase of GOT, compared to control, whereas MTX did not lead to alterations of this enzyme. The level of GTP was not significantly altered by the drugs compared to the control, although the venom tended to increase this marker. Markers of renal function, urea and creatinine, were measured (Fig. 5G,H, respectively); the treatments did not induce alteration in the serum levels of these markers.
PnV did not change total macrophages from spleen, but decreased macrophages expressing ifn-γ and IL-10. The venom increased spleen neutrophils and natural killer (NK) cells. The percentage of total macrophages (CD11b and F4/80 positive cells) isolated from the spleen were not altered by the treatment with venom, in comparison to the control group (data not shown). On the other hand, the percentage of macrophages (CD11b and F4/80 positive) expressing IFN-γ (activated/proinflammatory) (Fig. 6A-D) was strongly decreased by PnV therapy, compared to control. Interestingly, a similar result was observed for IL-10 expressing macrophages (suppressor/anti-inflammatory) (Fig. 6E-H). On the other hand, MTX had the opposite effect, significantly increasing macrophages in both profiles. Figure 7 shows that the treatment with PnV did not alter the percentage of neutrophils, compared to control (histograms A-C, and graph D); however, the MFI (Mean Fluorescence Intensity) and cytospin (Fig. 7E,F, respectively) revelated that PnV induced a significant increase in neutrophils compared to control. Analysis of NK cells (NK1.1 positive) showed that PnV increased the percentage cells, compared to control (Fig. 7F-H). MTX did not change neutrophils and NK cells from spleen in comparison to untreated group.

Animal
Tumor weight (mg) Tumor volume (mm 3 ) www.nature.com/scientificreports www.nature.com/scientificreports/ treatment with pnV increased the number of tumor associated macrophages (tAMs). Analysis of Iba-1 positive cells (a macrophage marker) within and around the tumor mass revealed that in the control tumor there were no macrophages (Fig. 8A-C). However, in the PnV treated mice, a large number of positive cells was present in the surrounding tissue, in the periphery of the tumor and within the tumor mass (Fig. 8D-F).
In vitro macrophages were activated by pnV. Fluorescent bioparticle phagocytosis assay (Fig. 9) showed that venom-treated macrophages (panels C, F and I) were more activated than untreated cells (panels A, D and G); LPS, used as a canonic positive control of macrophages activation to M1 profile, also induced increased  www.nature.com/scientificreports www.nature.com/scientificreports/ phagocytosis (panels B, E and H), however cells treated with PnV were even more active (See graph J in Fig. 9). Figure 9 shows Iba-1 labeled cells (panels A-C), fluorescent bioparticles (panels D-F) and Iba-1/bioparticles/ DAPI merged images (G-I). ELISA measures (graphs K and L in Fig. 9) demonstrated that while LPS induced increased TNF-alpha and IL-10 release, as expected, PnV did not change these cytokines.  www.nature.com/scientificreports www.nature.com/scientificreports/ of 0,1 µg/mL (Fig. 10B) and showed that only SF1 and SF12 had no effected on the viability of cells, comparing to control. Nine subfraction, SF2-SF11, demonstrated anti-tumor effect, being the SF6, SF10 and SF11 the most effective comparing to control cells.

Discussion
Recently, our group showed that P. nigriventer spider venom is cytotoxic to tumor cells in vitro, especially for NG97 GB cell line 7,14 . As follows, in the present work we aimed to investigate whether the systemic administration of the venom would alter the development of xenogeneic GB (NG97) implanted in mice. Females were used because the tumor development was more uniform than in males. The reason for this is unclear. The dose of PnV was chosen after preliminary tests, where other doses (850, 500, 200, 100 and 50 µg/Kg) were tested. Previous studies have shown that 850 µg/Kg was a sublethal dose in rats, however promoting several signs of intoxication (limb paralysis, convulsion, tremors, sialorrhea, sometimes death). For this study, the highest dose among the five   www.nature.com/scientificreports www.nature.com/scientificreports/ tested, which did not cause any clinical signs of intoxication in any animal, was chosen (100 µg/Kg). In addition, methotrexate (MTX) is a chemotherapeutic drug used to treat various types of cancer 22 ; Although not used for GB because the drug does not cross the BBB 23 , it is a classic chemotherapy that induces immunosuppression. Therefore, in this work, MTX was used as a control only to compare with the effects of PnV in relation to the effects on the immune cells.
The results demonstrated that PnV chemotherapy arrested tumor development. In addition, PnV tumors did not uptake detectable amounts of 18 F-FDG, suggesting impairment of the metabolism of tumor cells. High glucose uptake is a hallmark of cancer; FDG uptake was thought to indicate the aggressiveness of tumors and SUV is a well-known measure for FDG uptake in PET/CT 24 . Therefore, the lower uptake of FDG may indicate that the tumors of PnV treated animals were less aggressive compared to control.
It is relevant to report that in this study only one animal treated with PnV, instead of developing a tumor in the back (site of inoculation of glioma cells), developed a tumor mass in the brain. This was a unique case and the cause is unclear. However, the possibility that this may have happened because PnV is able to permeabilize the BBB has to be considered for future therapeutic use of molecules obtained from the venom, since metastasis can be facilitated by the opening of the barrier. In addition, previous studies have shown that, although the venom promotes BBB breakdown in specific regions, changes in cell morphology or necrosis were absent in the central nervous tissue 25 . On the other hand, PnV stimulates neurons and glia 13,26 , which may be related to the seizures observed in envenomed rats and humans. An important observation is that, with the dose used in this study, the mice did not present convulsion or any signs of intoxication. Nevertheless, venom is a mixture of molecular entities, and that with antineoplastic effects are hopefully to be different from those with target in the BBB, neuron and glia; therefore, when the molecules have been isolated, these aspects have to be examined and the effects are expected to be more specific towards cancer cells. www.nature.com/scientificreports www.nature.com/scientificreports/ Along with decreased tumor development, a better prognosis was observed in mice treated with PnV; the animals presented lower mortality in relation to the control and better recovery of weight. Nevertheless, PnV administration increased GOT levels in the serum, a marker of hepatic dysfunction. As same as before, we expect that purification of tumor active molecules may abolish hepatotoxic effect.
Histopathology showed that animals from control group had polymorphic hypercellular tumors, as expected. Other typical features were also present in this group, such as vascular proliferation, necrosis (with tumor cell palisading) and more than one cell type 27,28 . However, the majority of the tissue area in the PnV tumor was necrotic, lacking the histopathological characteristics typical of GB. This may explain the low uptake of 18 F-FDG in PnV tumors.
To assess the systemic effects of venom, blood cells were analyzed, showing that PnV increased monocytes and did not change neutrophils. This effect is different from that observed in classical chemotherapies, which induce a decrease of immune cells. In the present study, MTX was used only to compare the effects of PnV with a classical immunosuppressor chemotherapeutic drug. MTX induced a neutropenia, as expected, while it did not affect monocytes. It is reported that MTX can affect hematopoiesis, causing leukopenia, anemia, and/or thrombocytopenia 29 . Corroborating with the literature, it was shown here that while PnV did not alter erythrocytes, hemoglobin and hematocrits levels, in animals treated with MTX all these parameters were reduced. On the other hand, while PnV did not change platelets count, MTX treatment increased. The literature often reports a decrease in platelets (thrombocytopenia) 22 induced by MTX; however, thrombocytosis developed due to the use of MTX or MTX combined with other drugs has already been described [30][31][32] .
Considering the alterations induced by PnV in the blood immune cells, we decided to examine the profile of the immune cells isolated from the spleen. Interestingly, PnV did not change total macrophages, while decreased activated (IFN-γ positive) and suppressive (IL-10 positive) cells. Although PnV have decreased the percentage of activated and suppressors macrophages isolated from the spleen, many macrophages were detected in the tumor of animals treated with the venom, whereas they were not observed in the control. These results suggest that PnV induced an increase in blood monocytes, and these cells were directed preferentially to the tumor. Hypoxia is a common characteristic of many solid tumors and limited oxygen diffusion generates a gradient of oxygen availability from the blood vessel to the interstitial space and may underlie macrophages recruitment, promoting cancer progression 33 . The macrophages can be recruited to the tumor in PnV-treated animals because the tumor is highly necrotic and the environment is probably very poor of oxygen.
Macrophages in the tumor are known as Tumor Associated Macrophages (TAMs); TAMs exist in a spectrum, exhibiting spatiotemporal plasticity in regard to the phenotype 34,35 . In a simplified way, these cells can be divided into two subtypes, M1 and M2. Canonically, M1 is associated with pro-inflammatory properties and has antitumor effects, while M2 is associated with anti-inflammatory properties and promotes tumor growth and resistance to therapy 36 . Nevertheless, increasing evidence suggests that both contribute to the tumorigenesis and tumor cell migration/metastasis 37 . TNF-alpha and IL-12 are cytokines able to characterize M1, whereas IL-10 and IL-4 are able to characterize M2 phenotype 35,38 . Considering that PnV induced a decrease in polarized macrophages, both activated (INF-γ positive) and suppressed (IL-10 positive), it is possible that the profile of PnV-macrophages are phenotypically different of typical TAMs (M1 and M2) and may be more effective in fighting the tumor without promoting tumorigenesis.
It is important to mention that RAG −/− mice do not have the complete immune response (lacking T lymphocytes) and the immunomodulatory effects of PnV on other systems need to be tested. Therefore, to confirm and better understand the effects of the venom, cultured macrophages generated from mice bone marrow were treated with PnV. The venom activated the cells, which became more phagocytic than control (untreated). LPS, used as a positive control of M1 induced profile 21 , also increased phagocytosis. On the other hand, while LPS enhanced TNF-α and IL-10 cytokines release, as expected 39 , PnV-phagocytic macrophages did not produce those pro-or anti-inflammatory cytokines, corroborating the in vivo findings. The PnV-activated macrophages profile seems to be different of M1 or M2 phenotype. The effects of PnV and its molecules in the immune system needs to be better investigated; experiments in immunocompetent mice and in a 3D spheroid coculture system are in course.
A previous report 40 showed that P/Q-and N-type voltage-gated calcium channels (VGCC) inhibitors PhTx3-3 and Phα1β, from PnV, have inhibitory effects on proliferation and viability and evoked cell death of glioma cell lines (M059J, U138MG and U-251MG). Furthermore, Phα1β caused significant reduction of tumor areas in vivo and this toxin also increased GFAP-activated astrocytes and Iba-1 positive microglia in the peritumoral region, showing a modulation of these immune cells, which corroborates current findings. It has been shown here that purified fractions and subfractions of the PnV have antitumor action, decreasing the viability of GB cells; therefore, it is probable that not only calcium channel inhibitors are active in GBs. It is important to mention that the cytotoxic effect of PnV and most SFs, although significant, was not very pronounced. Our hypothesis is that this effect is added to the immunological activation against the tumor, resulting in a synergistic therapeutic action, justifying the strong tumor reduction observed in this study in the in vivo model. We are isolating and characterizing the immunomodulatory and antitumor PnV molecules. In addition, components of the venom with anti-migratory effects in GB cells are being identified.
In conclusion, the venom was effective in impairing tumor development in a murine xenogeneic model. It has also been demonstrated for the first time that the venom has immunomodulatory effects, and this may be an essential mechanism of the antineoplastic role of the venom components. The effects of PnV on immune cells suggest that its components did not induce immunosuppression, like classical chemotherapeutic drugs. In addition, PnV treatment increased macrophages into and surround the tumor, however with a different profile of M1. Studies in progress are selecting the venom molecules with antineoplastic and immunomodulatory effects and trying to better understand their mechanisms. The identification and synthesis of antineoplastic/immunomodulatory molecules can lead to a new multitarget chemotherapy.