Abstract Bone metastases are a frequent complication of advanced breast cancer, against which immune checkpoint inhibitors are not effective. We previously identified that PD-1+ T cells in a mouse model of bone metastases are inactivated, potentially because of the expansion of PD-L1+ myeloid-derived suppressor cells (MDSC) in bones. 5-Fluorouracil (5-FU) can decrease MDSC in thymoma and colon cancer models. Thus, we aim to determine the efficacy of 5-FU in breast cancer bone metastases as well as its potential side effects.Balb/C mice were inoculated in the left cardiac ventricle with 4T1 breast cancer cells to cause bone metastases within 10 days. A treatment with 4 inoculations of 5-FU (50 mg/kg) prevented splenomegaly and the expansion of PMN- and M-MDSC in bones, as assessed by flow cytometry. Treatments with doses of 10 or 25 mg/kg were not as efficient against MDSC. Also, while these lower doses tended to increase T cell infiltration, 50 mg/kg decreased it, suggesting toxicity. Thus, we modulated the number of inoculations at 50 mg/kg. Two and three inoculations decreased the splenomegaly and histological damage, but also the total number of bone marrow cells. Flow cytometry confirmed these treatments decreased PMN-MDSC, while there was only a trend toward a decrease in M-MDSC in bone metastases. More interestingly, the numbers of CD4+ and CD8+ T cells were increased, especially naïve and central memory T cells. On radiographs, the bone destruction caused by cancer cells was also decreased. However, a single inoculation did not cause any long-lasting effects on MDSC and T cells, and the osteolysis area was not decreased. As 5-FU sharply decreased the number of bone marrow cells in bone metastases, we sought to assess potential side effects. Three inoculations of 5-FU in healthy mice decreased neutrophils, monocytes, B cells, and NK cells in bones 3 days after the last inoculation, while T cells were still increased. However, these effects were transient since a week later, the mice had recovered. We evaluated levels of hematopoietic stem cells (HSC) and myeloid and lymphocytic progenitor cells (MPC, LPC). Surprisingly, in these conditions, 5-FU transiently increased the number of HSC and LPC, which could explain the rapid recovery, while MPC remained stable. Cancer treatments can affect bones and increase the risk of fracture, so we assessed the bone 3D microarchitecture using microCT. Two and three inoculations of 5-FU did not cause any changes in the trabecular bone volume, or the thickness or number of the trabeculae. Surprisingly, histomorphometry analysis did not reveal any changes in the number of osteoclasts, the myeloid cells dedicated to bone resorption. Thus, 5-FU could be used to decrease MDSC and increase T cells in bone metastases, turning them into a hot microenvironment to increase the efficacy of immunotherapies in breast cancer patients. Citation Format: Galilea Chora Hernández, Paloma Almeida Luna, Kiomy Esparza Palomares, Erasmo Pérez Mendez, Samanta Jimenez, Ricardo Gonzalez Sanchez, Candice Internicola, Felipe Olvera, Pierrick G.J. Fournier. Modulation of the tumor microenvironment using 5-fluorouracil to increase T cell infiltration in breast cancer bone metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2840.

Snake venoms are complex biochemical phenotypes whose composition can diverge dramatically among taxa, yet the functional and biomedical consequences of such variation remain understudied for many lineages. Here, we characterized the functional activities of venom from multiple rattlesnake species distributed across insular and mainland localities in the Baja California Peninsula and Gulf of California. We quantified phospholipase A2 (PLA2), proteolytic, and fibrinogenolytic activities as well as median lethal doses (LD50) for 55 individuals across Crotalus enyo, C. mitchellii, C. pyrrhus sensu lato, and C. ruber. We also evaluated the neutralization efficacy of the commercial polyvalent antivenom Antivipmyn®. Venom functional activities differed significantly among species. Crotalus mitchellii expressed a neurotoxic phenotype characterized by the highest PLA2 activity, the lowest LD50 values, and minimal proteolysis. Crotalus pyrrhus and C. ruber expressed proteolytic phenotypes with high enzymatic activity and ubiquitous dual-chain fibrinogenolysis. Crotalus enyo exhibited intermediate functional activities with moderate enzymatic output and the most efficient antivenom neutralization among species. Although Antivipmyn® neutralized the lethal activity of all venoms, efficacy differed significantly among species in both mass-normalized (mgAV/mgV) and potency-scaled (LD50-equivalents per vial) metrics. Notably, insular and mainland populations within species showed qualitative variation in both functional activities and antivenom responses. These results establish that the focal species possess functionally distinct venom phenotypes with species-specific consequences for antivenom neutralization, underscoring the relevance of venom functional characterization for clinical management of envenomation across the Baja California region.

The Crotalus molossus complex consists of five to seven phylogenetically related lineages of black-tailed rattlesnakes widely distributed in Mexico. While previous studies have noted venom variation within specific lineages of the Crotalus molossus complex, a comprehensive characterization of interspecific and ontogenetic venom variations, their functional implications, and the neutralizing ability of the Mexican antivenom against these variants remains largely unexamined. Herein, using two proteomic approaches for five lineages (C. basiliscus, C. m. molossus, C. m. nigrescens, C. m. oaxacus, and C. ornatus) of the C. molossus complex we characterized the number of toxins and their relative abundance in the venom of individuals of varying sizes. All five lineages undergo ontogenetic venom composition shifts associated with snake length. However, the pattern of ontogenetic shifts varied among lineages. In some lineages, these shifts led to significant differences in proteolytic, phospholipase A2, and fibrinogenolytic activities. Venom in smaller C. basiliscus, C. m. nigrescens, and C. m. oaxacus individuals had lower LD50 values (more lethal) in mice. Whereas the venom lethality of C. m. nigrescens (both juvenile and adult) and C. m. oaxacus (adult) was several times higher in a mammalian (mouse) model than in a reptilian (iguana) model. Antivipmyn® showed different neutralizing potencies toward venom pools. Overall, our results indicated that even among closely related rattlesnake lineages, venom phenotypes may vary greatly, impacting their function and the efficacy of antivenom neutralization.

Triple-negative breast cancer (TNBC) is an aggressive subtype of cancer characterized by limited therapeutic options and a propensity to form metastasis, particularly to bone. Given the mortality and morbidity linked to TNBC, the advancement of novel and effective therapies is essential. This work studies the potential of an adenosine derivative compound (IFC-305) as a therapeutic agent for breast cancer bone metastasis. It focuses on its effects on bone metastasis, including cell viability, migration, cell cycle, and apoptosis in vitro. In vitro assays demonstrated that IFC-305 significantly reduced TNBC cell viability and migration in a dose-dependent manner. Additionally, it promoted cell cycle arrest and decreased the proportion of cells in the S phase, although it did not induce apoptosis in 4T1 cells. Furthermore, in vivo experiments using a murine model of bone metastasis revealed that treatment with IFC-305 attenuated osteolytic lesions associated with breast cancer metastasis. Importantly, our findings highlight an interaction between this adenosine derivative and the TGF-β signaling pathway. The derivative compound effectively inhibited SMAD2/3 phosphorylation, a key mediator of TGF-β signaling pathway involved in cell growth, differentiation, migration and metastatic progression. Besides, IFC-305 reduced TGF-β regulated PMPEA1 expression, a gene associated with cancer stemness and tumor invasion. These findings provide evidence for the therapeutic potential of the IFC-305 in treating metastatic breast cancer in the bone and warrant further investigation to fully elucidate its mechanism of action and evaluate its clinical translatability.

Crotalus culminatus is a medically significant species of rattlesnake in Mexico [1]. While the proteomic composition of its venom has been previously reported for both juvenile and adult specimens, there has been limited research into its functional properties, with only a few studies, including one focusing on coagulotoxicity mechanisms. In this study, we aimed to compare the biochemical and biological activities of the venom of juvenile and adult snakes. Additionally, we assessed antibody production using the venoms of juveniles and adults as immunogens in rabbits. Our findings reveal lethality and proteolytic activity differences between the venoms of juveniles and adults. Notably, juvenile venoms exhibited high proportions of crotamine, while adult venoms displayed a reduction of this component. A commercially available antivenom demonstrated effective neutralization of lethality of both juvenile and adult venoms in mice. However, it failed to neutralize the paralytic activity induced by crotamine, which, in contrast, was successfully inhibited by antibodies obtained from hyperimmunized rabbits. These results suggest the potential inclusion of C. culminatus venom from juveniles in commercial antivenom immunization schemes to generate antibodies targeting this small myotoxin.

Abstract Bone metastases are a highly debilitating complication in more than 70% of patients with advanced breast and prostate cancer. Currently approved treatments fail to cure bone metastases or increase patient survival. Although immunotherapies can cause a durable response in some patients, it seems that their efficacy is limited in bone metastases, which could be due to a cold metastatic microenvironment. Our aim was to identify immunosuppressive factors in bone metastases and target them using a drug-repositioning type approach. We used a syngeneic model in which 4T1 triple-negative breast cancer cells derived from a Balb/C mouse are inoculated in the left cardiac ventricle of Balb/C mice to cause osteolytic bone metastases. Using flow cytometry, we found that the bone marrow of mice with 4T1 bone metastases is enriched in large cells with a high SSC index that could be myeloid-derived suppressor cells. We confirmed that the inoculation of 4T1 cells increased the amount of CD11b+ Ly6G+ Ly6Clo ROShi and CD11b+ Ly6G- Ly6Chi NOhi cells. Both cell types were isolated from bone metastases and prevented the proliferation of CD8+ T cells ex vivo, confirming they were polymorphonuclear- (PMN-) and monocytic-MDSCs (M-MDSCs), respectively. In addition, both types could differentiate into osteoclasts that cause bone loss. Reviewing the current literature, we found that FDA-approved sildenafil (a PDE-5 inhibitor), zoledronic acid (a bone resorption inhibitor), and 5-fluorouracil (5-FU, a chemotherapeutic agent) can decrease MDSCs in other cancer models. However, sildenafil (20mg/kg, i.p., every 2 days) and zoledronic acid (25µg/kg, s.c., 3x/week) did not decrease the amount of MDSCs in 4T1 bone metastases. 5-FU (i.p., 4 inoculations) at 50mg/kg prevented the 4T1-induced splenomegaly and the increase of PMN- and M-MDSCs but seemed to decrease the number of T cells in bone metastases. Thus, we tested the efficacy of a lower number of inoculations. A single inoculation of 5-FU was not efficient, while 2 or 3 inoculations of 5-FU (50mg/kg) prevented the splenomegaly and the histological damage due to extramedullary hematopoiesis in the spleen. In 4T1 bone metastases, there was a decrease of PMN-MDSCs and an increase of the number of CD4+ and CD8+ T cells. On radiographs, 2 or 3 inoculations of 5-FU (50mg/kg) significantly decreased the area of the osteolytic lesions caused by 4T1 cells (-57% and -60%, respectively, compared to placebo, P<0.005). In conclusion, our results show that functional PMN- and M-MDSCs can increase and accumulate in the microenvironment of breast cancer bone metastases and that an optimized treatment with 5-FU can prevent this expansion of MDSC, increasing T cell infiltration in bone metastases and decrease the associated bone lesions. Treatment with 5-FU could then contribute to turning bone metastases from a cold to a hot microenvironment and increase the efficacy of immunotherapies in patients. Citation Format: Pierrick G.J. Fournier, Paloma Almeida Luna, Danna Arellano, Samanta Jiménez, Felipe Olvera, Patricia Juárez. 5-Fluorouracil decreases MDSC and the development of osteolytic bone metastases from breast cancer in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5281.

Background: Scorpion neurotoxins such as those that modify the mammalian voltage-gated sodium ion channels (Nav) are the main responsible for scorpion envenomation. Their neutralization is crucial in the production of antivenoms against scorpion stings. Methods: In the present study, two in silico designed genes - one that codes for a native neurotoxin from the venom of the Anatolian scorpion Androctonus crassicauda, named Acra 4 - and another non-native toxin - named consensus scorpion toxin (SccTx) obtained from the alignment of the primary structures of the most toxic neurotoxins from the Middle Eastern and North African scorpions - were recombinantly expressed in E. coli Origami. Results: Following bacterial expression, the two expressed neurotoxins, hereafter named HisrAcra4 and HisrSccTx, were obtained from inclusion bodies. Both recombinant neurotoxins were obtained in multiple Cys-Cys isoforms. After refolding, the active protein fractions were identified with molecular masses of 8,947.6 and 9,989.1 Da for HisrAcra4 and HisrSccTx, respectively, which agreed with their expected theoretical masses. HisrAcra4 and HisrSccTx were used as antigens to immunize two groups of rabbits, to produce either anti-HisrAcra4 or anti-HisrSccTx serum antibodies, which in turn could recognize and neutralize neurotoxins from venoms of scorpion species from the Middle East and North Africa. The antibodies obtained from rabbits neutralized the 3LD50 of Androctonus australis, Leiurus quinquestriatus hebraeus and Buthus occitanus venoms, but they did not neutralize A. crassicauda and A. mauritanicus venoms. In addition, the anti-HisrAcra4 antibodies did not neutralize any of the five scorpion venoms tested. However, an antibody blend of anti-HisrAcra4 and anti-HisrSccTx was able to neutralize A. crassicauda and A. mauritanicus venoms. Conclusions: Two recombinant Nav neurotoxins, from different peptide families, were used as antigens to generate IgGs for neutralizing scorpion venoms of species from the Middle East and North Africa.

Here we report, for the first time, a natural hybrid between Crotalus atrox and C. mictlantecuhtli based on intermediate characteristics of the external morphology and venom. Morphologically, the individual had characteristics of both parent species. The hybrid's venom exhibited an intermediate composition including the presence of crotoxin which has never been documented in C. atrox but is well documented in C. mictlantecuhtli. The hybrid's venom was highly toxic and showed an intermediate proteolytic activity between the parental species. The two Mexican antivenoms were able to neutralize the hybrid's venom's lethality.

Alpha-latrotoxin (aLTx) is the component responsible for causing the pathophysiology in patients bitten by spiders from the genus Latrodectus, commonly known as black widow spiders. The current antivenom used to treat these envenomations in Mexico is produced using the venom of thousands of spiders, obtained through electrical stimulation. This work aimed to produce this protein as well as two of its fragments in a bacterial model, to evaluate their use as immunogens to produce neutralizing hyperimmune sera, in rabbits. aLTx is a 130 kDa protein which has not yet been obtained in a soluble active form using bacterial models. In the present work, aLTx and two of its fragments, ankyrin domain and amino terminal domain (LTxAnk and LTxNT) were produced in bacteria and solubilized from inclusion bodies using N-lauroyl sarcosine. These three proteins were used for hyperimmunization in order to evaluate their potential as immunogens for the production of neutralizing hyperimmune sera against the complete venom of Latrodectus mactans. The hyperimmune sera obtained using the complete aLTx as well as the LTxNT, was capable of pre-venting death of mice envenomated with 3 LD50s of venom, both in preincubation and rescue experiments. Conversely, the serum obtained using the LTxAnk fragment, generated only partial protection and a delay in the time of death, even with a maximum dose of 450 mL. We therefore conclude that the produced proteins show great potential for their use as immunogens and should be further tested in large animals, such as horses. (C) 2022 Published by Elsevier B.V.

Crotamine is a paralyzing toxin (MW: ~5 kDa) found in different proportions in some rattlesnake venoms (up to 62%). Mexican pit viper antivenoms have shown low immunoreactivity against crotamine, which is an urgent quality to be improved. The objective of this work was to evaluate the ability of a novel recombinant fusion protein composed of sphingomyelinase D and crotamine, and two whole venoms from Crotalus molossus nigrescens and C. oreganus helleri to produce neutralizing antibodies against crotamine. These immunogens were separately used for immunization procedures in rabbits. Then, we generated three experimental antivenoms to test their cross-reactivity via western-blot against crotamine from 7 species (C. m. nigrescens, C. o. helleri, C. durissus terrificus, C. scutulatus salvini, C. basiliscus, C. culminatus and C. tzabcan). We also performed pre-incubation neutralization experiments in mice to measure the neutralizing potency of each antivenom against crotamine induced hind limb paralysis. Our antivenoms showed broad recognition across crotamine from most of the tested species. Also, neutralization against crotamine paralysis symptom was successfully achieved by our three antivenoms, albeit with different efficiencies. Our results highlight the use of crotamine enriched venoms and our novel recombinant fusion protein as promising immunogens to improve the neutralizing potency against crotamine for the improvement of Mexican antivenoms.

Background: Micrurus venoms contain two main groups of toxic protein components: short-chain alpha-neurotoxins (SNtx) and phospholipases type A(2) (PLA(2)). In North America, generally, the Micrurus venoms have low abundance of SNtx compared to that of PLA(2)s; however, both are highly toxic to mammals, and consequently both can play a major role in the envenomation processes. Concerning the commercial horse-derived antivenoms against Micrurus from the North America region, they contain a relatively large amount of antibodies against PLA(2)s, and a low content of antibodies against short chain alpha-neurotoxins. This is mainly due to the lower relative abundance of SNtxs, and also to its poor immunogenicity due to their size and nature. Hence, Micrurus antivenoms made in North America usually present low neutralizing capacity towards Micrurus venoms whose lethality depend largely on short chain alpha-neurotoxins, such as South American Micrurus species. Methods: Horses were hyperimmunized with either the venom of M. tener (PLA(2)-predominant) or a recombinant short-chain consensus alpha-neurotoxin (ScNtx). Then, the combination of the two monospecific horse antibodies (anti-M. tener and anti-ScNtx) was used to test their efficacy against eleven Micrurus venoms. Results: The blend of anti-M. tener and anti-ScNtx antibodies had a better capacity to neutralize the lethality of diverse species from North, Central and South American Micrurus venoms. The antibodies combination neutralized both the ScNtx and ten out of eleven Micrurus venom tested, and particularly, it neutralized the venoms of M. distans and M. laticollaris that were neither neutralized by monospecific anti-M. tener nor anti-ScNtx. Conclusions: These results provide a proof-of-principle for using recombinant immunogens to enrich poor or even non-neutralizing antisera against elapid venoms containing short chain alpha-neurotoxins to develop antivenoms with higher effectiveness and broader neutralizing capacity. (C) 2020 Elsevier Ltd. All rights reserved.

Bone metastases is a frequent (>75%) complication of advanced breast cancer (BCa). BCa cells in bone are supported by cytokines released during the osteoclastic resorption, and remain untreatable. T cells during immunotherapy could turn against BCa cells in bone but could also increase osteoclasts and bone metastases. Comparing the development of bone metastases from 4T1 BCa cells between Balb/C and SCID or T cell-depleted mice, the absence of T cells decreased bone metastases (−72% and −38%, respectively), while orthotopic tumors were increased. Histology confirmed that the osteoclast number was decreased at the tumor-bone interface of mice lacking T cells. Ex vivo addition of non-activated T cells increased osteoclast formation, and flow cytometry confirmed that >85% of T cells in bone metastases were not expressing activation markers. In sharp contrast, activated T cells were potent inhibitors of osteoclast formation, which could be beneficial for the treatment of patients. Although activated T cells expressed the anti-osteoclastic Ifng and Il4, their effect was not reversed by neutralizing antibodies, or due to a bystander effect by cytotoxic T cells. Thus, we sought to activate T cells of bone metastases. However, it was not possible to activate T cells in bone marrow cultures ex vivo. This could be due to an increase of MDSCs in 4T1 bone metastases, including M-MDSCs that were PD-L1+ (>85%) and could suppress PD-1+ T cells (>70%) in bone. Our results suggest that while T cells under the influence of the bone microenvironment promote BCa bone metastases, immunotherapy-activated T cells could suppress bone resorption and eliminate BCa cells in patients with bone metastases.

Breast cancer (BCa) cells disseminating to the bone can remain dormant and resistant to treatments for many years until relapsing as bone metastases. The tyrosine kinase receptor TIE2 induces the dormancy of hematopoietic stem cells, and could also induce the dormancy of BCa cells. However, TIE2 is also a target for anti-angiogenic treatments in ongoing clinical trials, and its inhibition could then restart the proliferation of dormant BCa cells in bone. In this study, we used a combination of patient data, in vitro, and in vivo models to investigate the effect of TIE2 in the dormancy of bone metastases. In BCa patients, we found that a higher TIE2 expression is associated with an increased time to metastases and survival. In vitro, TIE2 decreased cell proliferation as it increased the expression of cyclin-dependent kinase inhibitors CDKN1A and CDKN1B and arrested cells in the G0/G1 phase. Expression of TIE2 also increased the resistance to the chemotherapeutic 5-Fluorouracil. In mice, TIE2 expression reduced tumor growth and the formation of osteolytic bone metastasis. Together, these results show that TIE2 is sufficient to induce dormancy in vitro and in vivo, and could be a useful prognostic marker for patients. Our data also suggest being cautious when using TIE2 inhibitors in the clinic, as they could awaken dormant disseminated tumor cells.

Abstract Bone metastases are a frequent complication in patients with advanced breast cancer and remain incurable. Thus, we need to explore new options for their treatment. Immunotherapy for the treatment of cancer is promising but remains untested for bone metastases. A potential limitation for such use is the ability of T cells to activate osteoclasts and bone resorption that releases pro-metastatic growth factors. Thus, we aim to characterize the effect of T cells in bone metastases. When inoculated in Balb/C SCID mice that lack T and B cells or in T cell-depleted Balb/C mice, 4T1 cells caused 72% and 38% less bone metastases, respectively (p<0.05), when compared to control mice. Histology confirmed that mice with a functional immune system have an increased number of osteoclasts at the tumor/bone interface. Consistently, the addition of mouse T cells to bone marrow culture ex vivo increased the formation of osteoclasts. However, this behavior was limited to non-activated T cells. Upon activation with antibodies against CD3 and CD28, T cells inhibited the formation of osteoclasts in a dose-dependent manner. This anti-osteoclastic effect was not due to a cytotoxic effect of T cells, and was consistent with decreased levels of the pro-osteoclastic Rankl and increased expression of anti-osteoclastic Ifng and Il4. When measured in vivo, Ifng was not detected, and levels of pro-osteoclastic Rankl and Tnfa were higher in T cells from 4T1 bone metastases compared to splenic T cells, suggesting that T cells in the bone marrow are not activated. Flow cytometry confirmed that only up to 15% of T cells in bone metastases expressed the activation marker CD69. Interestingly, T cells in the bone marrow could not be further activated ex vivo, unlike T cells from the spleen. This could be due to increased levels of the immunosuppressive monocytic (CD11b+Ly6C+Ly6G−) and polymorphonuclear (CD11b+Ly6G+Ly6C−) Myeloid Derived Suppressor Cells (MDSC) in bone metastases. To activate T cells in vivo, we attempted to target MDSCs using the PDE-5 inhibitor, Sildenafil, in combination with zoledronate, an FDA-approved bone resorption inhibitor. Still, there were no effects on MDSCs or the development of bone metastases. Therefore, we sought other therapeutic targets, such as immune checkpoints. Although only ~27% of T cells in 4T1 bone metastases were CTLA-4+, >70% of T cells were PD-1+, and ~80% of monocytic-MDSC were PD-L1+, which could trigger T cell suppression in bone. In conclusion, we found that the bone metastasis microenvironment suppresses T cells, making them pro-osteoclastic and increases the development of bone metastases. We also found that activation of T cells prevented the formation of osteoclasts. Therefore, the activation of T cells using immunotherapy, such as immune checkpoint inhibitors (anti-PD-1 or anti-PD-L1 antibodies), could be used for the treatment of patients with bone metastases. Citation Format: Danna L. Arellano, Juan A. Corral-Avila, Florian Drescher, Andrea Verdugo-Meza, Samanta Jimenez, Felipe Olvera-Rodriguez, Patricia Juarez, Pierrick Fournier. Bone microenvironment-suppressed T cells increase osteoclast formation and breast cancer bone metastases [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6199.

Antivenoms are fundamental in the therapy for snakebites. In elapid venoms, there are toxins, e.g. short-chain α-neurotoxins, which are quite abundant, highly toxic, and consequently play a major role in envenomation processes. The core problem is that such α-neurotoxins are weakly immunogenic, and many current elapid antivenoms show low reactivity towards them. We have previously developed a recombinant consensus short-chain α-neurotoxin (ScNtx) based on sequences from the most lethal elapid venoms from America, Africa, Asia, and Oceania. Here we report that an antivenom generated by immunizing horses with ScNtx can successfully neutralize the lethality of pure recombinant and native short-chain α-neurotoxins, as well as whole neurotoxic elapid venoms from diverse genera such as Micrurus , Dendroaspis , Naja , Walterinnesia , Ophiophagus and Hydrophis . These results provide a proof-of-principle for using recombinant proteins with rationally designed consensus sequences as universal immunogens for developing next-generation antivenoms with higher effectiveness and broader neutralizing capacity.

Abstract After arriving in bone, cancer cells can enter a dormancy state that can last for years, during which they are resistant to treatment. The bone microenvironment induces dormancy via mechanisms similar to the ones of hematopoietic stem cells (HSC). The angiopoietin receptor, TIE2, induces the dormancy of HSC and some cancer cells and is a target for anti-angiogenic treatments. However, its inhibition may restart the proliferation of cancer cells, posing the risk of bone metastasis growth, while increasing the sensitivity of cancer cells to chemotherapy. Thus, we aim to characterize the role of TIE2 in dormancy of bone metastases. We found that higher levels of Tie2 in the primary tumor of breast cancer (BCa) patients are associated with a good prognosis in multiple cohorts. However, expression of Tie2 protein or mRNA was either very low or undetected in cultured BCa and prostate cancer (PCa) cell lines. BCa and PCa cells transduced to express TIE2 constitutively showed a gradual loss of TIE2+ cells, despite continuous antibiotic selection. Congruently, in 5 different patient datasets, Tie2 expression is significantly lower in invasive BCa compared to healthy breast tissue. These results suggest that there is no expression of TIE2 in growing cancer cells. In MCF-7 TIE2tet BCa cells conditional TIE2 expression with a Tet-On system caused a significant decrease of cell proliferation and the percentage of cells in S and G2/M phase but not in MCF-7 GFPtet control cells. Expression of TIE2, but not GFP, significantly decreased mRNA levels of the proliferation marker Ki67 while increasing the dormancy markers p21 and p27. Additionally, TIE2 expression increased the resistance to the chemotherapeutic 5-Fluorouracil. 4T1 Tie2tet cells inoculated in the mammary fat pads of Balb/C mice receiving Dox showed significantly reduced tumor volume and weight of excised tumors compared to control mice, suggesting TIE2 caused a growth reduction. When intracardiacally inoculated, 4T1 Tie2tet cells caused bone metastases regardless of TIE2 expression. However, the osteolysis area was significantly decreased when TIE2 was expressed, indicating a delayed bone metastasis development. To reverse dormancy and increase chemotherapy sensitivity, we selected a shark antibody (vNAR) that binds to TIE2 using a synthetic library and phage display. In silico modeling indicated this clone could bind to the FN2 domain and inhibit the multimerization of TIE2, which could prevent signaling. Overall, we found that expression of TIE2 decreases BCa cell growth in vitro and in vivo, and their sensitivity to 5-Fluorouracil, while increasing the expression of dormancy marker in vitro, and the time to the development of metastasis and survival of BCa patients. Our results are then consistent with TIE2 inducing dormancy of cancer cells. Thus, neutralizing TIE2 with a vNAR in combination with cancer therapy could be used for the treatment of patients at risk of bone metastases. Citation Format: Florian Drescher, Salvador Dueñas, Felipe Olvera-Rodriguez, Nicolás Serafín Higuera, Samanta Jiménez Flores, Patricia Juarez, Alexei Licea-Navarro, Pierrick G. Fournier. Characterization of TIE2 function in cancer cell dormancy and bone metastases [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2651.

This work evaluated the feasibility of using toxoids obtained by gamma radiation in the production of antivenoms in small and large animals. Mixtures of African snake venoms from viperids or elapids were used. The viperid mixture contained the crude venom of five species of the genera Echis and Bitis, while the elapid mixture contained the crude venom of six species of the genera Naja and Dendroaspis. The viperid mixture had an LD50 of 1.25 mg/kg in mice, and the elapid mixture had an LD50 of 0.46 mg/kg. Both viper and elapid aqueous mixtures were subjected to Cobalt-60 gamma irradiation in three physical states: lyophilized, frozen and liquid. Radiation doses ranged from 0.5 to 100 kGy. The LD50s of the lyophilized and frozen mixtures of both viperid and elapid mixtures remained unaltered with radiation doses as high as 100 kGy; nevertheless, in the liquid state, doses of 3.5 and 5.5 kGy reduced the venom toxicity of both the viperid and elapid mixtures to 7.25 mg/kg and 1.74 mg/kg; less toxic by factors of 5.8 and 3.8, respectively. Groups of four rabbits and three horses were immunized with either irradiated or non-irradiated mixtures. In vitro and in vivo analysis of the rabbit and horse sera revealed that neutralizing antibodies were produced against both irradiated (toxoids) and native venom mixtures. None of the animals used in this study, either immunized with native venom or toxoids, developed severe local effects due to the application of venoms mixtures. Gamma-irradiated detoxified venoms mixtures, under well-controlled and studied conditions, could be a practical alternative for the production of polyvalent equine serum with high neutralization potency against snake venoms.

This work reports a new approach to clone active Neopladine 2 (Neo2), an anticancer peptide from Tityus discrepans scorpion venom. RNA was extracted from the venom gland and cDNA was obtained by RTPCR using complementary primers for the nucleotide sequence coding native Neo2 (nNeo2) N-terminal. Deduced amino acid sequence shows that Neo2 is comprises by 245 residues. The best conditions to achieve active Neo2 expression in Escherichia coli were 16 degrees C for 20-24 h. Active recombinant Neo2 (rNeo2) was obtained as inclusion bodies (IBs) with a yield of 3.5 mg/L culture. rNeo2 purification by HPLC revealed eleven isoforms with differences in polarity and activity. Only rNeo2a and rNeo2k isoforms induces cancer cells death like nNeo2. rNeo2a, rNeo2g and rNeo2j were the most interesting isoforms because their apoptosis/necrosis (A/N) index and their yield. Cloned isoforms have attractive properties because some of them had higher activity than the native protein, which can be exploited for biotechnological and biomedical applications. The in silico Neo2 3D structure predicted a globular protein, with five alpha-helixes and four beta-sheet maintained by 4 sulfide pairing, highly polar, with charged amino acids in its surface and two faces with different electrostatic potentials, suggesting an important dipolar moment. (C) 2016 Published by Elsevier Ltd.