Intratumoral injection of a novel TLR4 agonist elicits infiltration by immune effector cells in a canine soft tissue sarcoma: An Immunologic Case Study

Intratumoral injection of a novel TLR4 agonist elicits infiltration by immune effector cells in a canine soft tissue sarcoma: An Immunologic Case Study | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Case Report Intratumoral injection of a novel TLR4 agonist elicits infiltration by immune effector cells in a canine soft tissue sarcoma: An Immunologic Case Study Stuart C Helfand, Mariano Carossino, Jiho Kim, David R McMonigle, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8982168/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 14 You are reading this latest preprint version Abstract Background Immunostimulatory adjuvants used in vaccines to protect against infectious disease have demonstrated efficacy in stimulating anti-cancer immunity. The most commercially advanced ones activate Toll-Like Receptor-4 (TLR4), a transmembrane signaling molecule expressed by macrophages and dendritic cells triggering innate immune responses. Lipopolysaccharide, the first identified TLR4 agonist, induces toxic, unregulated immune activation. Mimetics of monophosphoryl lipid A, the stimulatory component in lipopolysaccharide, reduced immunotoxicity while retaining immunostimulatory properties. Intratumoral injection of formulated TLR4 agonists can stimulate in situ antitumor immune responses by recruitment of immune cells and production of inflammatory cytokines that exert antitumor effects via a variety of mechanisms – including direct cancer cell death and recruitment of effector cells. Human clinical cancer trials have shown efficacy - both locally and through abscopal effects employing this approach. Methods We hypothesized that injection of a novel TLR4 agonist, EmT4™, into a canine soft tissue sarcoma (STS) could alter the tumor microenvironment by attracting and activating immune cells in situ . With the dog owner’s interest and written consent, a 3-cm soft tissue mass on the right forelimb of an 8-year-old female spayed Boston terrier received two intratumoral injections of EmT4™, two weeks apart. There was transient lethargy on the day of the first injection that resolved within hours. The tumor was excised 4 weeks after the second injection. Histopathology, immunohistochemistry, and in situ RNA hybridization were utilized to explore immune cell populations in the tumor microenvironment. Results Histopathology revealed grade 2 STS with large numbers of densely packed perivascular immune cells disseminated within the tumor. Immunohistochemistry for immune cell markers showed heterogeneous positive staining within cell clusters – CD3 (25%), CD20 (57%), FOXP3 (8%), CD204 (5%), and Iba-1 (36%). In situ hybridization performed on serial STS sections with RNAscope™ identified transcripts for CD4 (29%), TNF-α (24%), CD8 (3.2%), and interferon-g (1.3%) in lymphocyte clusters. Conclusions EmT4™ may have elicited an innate immune response that attracted and activated immune effector cells intratumorally. Clinical circumstances prevented acquisition of a pre-EmT4™ biopsy hampering a definitive conclusion although cell infiltrates observed are unusual in canine STS. This case is foundational for continued EmT4™ investigations for canine cancer immunotherapy. toll-like receptor 4 lymphocytes monophosphoryl lipid A lipopolysaccharide mimetic immunogenic cell death inflammatory cytokines immunostimulatory Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background A major goal of cancer immunotherapy is to modify the tumor microenvironment (TME) so that conditions favor a tumor-specific immune response. Multiple mechanisms within the TME prevent immune effector cells from attacking the cancer even if the cells manage to penetrate the tumor parenchyma. Some tumors are devoid of effector cells because they have a low mutational burden and/or express fewer tumor antigens. The lack of tumor neoantigens can reduce influx of immune effector cells into the TME, thereby preventing a tumor-specific immune response. Once activated, the immune system can be highly effective against cancer as demonstrated by remarkable results observed when using check point inhibitors and other therapeutic monoclonal antibodies. One strategy to reprogram the TME is by directly injecting immune stimulants within the tumor architecture to induce immunologic hotspots in situ . 1 In this report, intratumoral (IT) immune infiltration is described in a canine soft tissue sarcoma (STS) following intralesional administration of an innate immune system activator. We used the adjuvant EmT4™ which contains a synthetic mimetic of monophosphoryl lipid A (MPLA) called 3D (6-acyl)-PHAD, the core structure of bacterial lipopolysaccharide (LPS). It targets and activates Toll-like receptor 4 (TLR4) without the toxicity associated with LPS. 2 Triggering TLR4 and its signaling pathways can lead to potent innate immune responses. TLR4 is evolutionarily conserved across mammalian species indicating its importance in immune responses - especially in response to gram negative bacterial LPS. For example, Leptospira vaccines for dogs trigger identical immune responses in multiple species through TLR activation. Canine monocyte-derived dendritic cells were used in the study to verify canine expression and activation by MPLA. 3 Since MPLA activates TLR4 in the dog and the active ingredient of EmT4™ is a synthetic mimetic of MPLA, we hypothesized that canine immune cells would efficiently respond to injection of this immune stimulant. 4,5 This activator is modeled on adjuvants used in vaccines that turn on robust immune responses by binding pattern recognition receptors or TLRs on dendritic cells and macrophages. Activation of the TLR4 signaling pathway results in induction of pro-inflammatory genes - including IL-1β, TNF-α, and type 1 interferon - which in turn upregulate TLR4 expression and immune cell recruitment. Chemokines and cytokines induced by TLR4 activation can mediate immunogenic tumor cell death. Tumor antigens phagocytosed and processed by activated dendritic cells and macrophages are presented to T-cells within the tumor and local draining lymph nodes. 6 T cells that traffic to draining lymph nodes can expand and are then capable of returning to the tumor en masse as well as circulating systemically and mediating abscopal cytotoxicity at metastatic sites (Fig. 1 ). 7 The steps in this process recapitulate those following vaccination with adjuvants for infectious diseases and can be considered in situ autovaccination against a cancer that facilitates polyclonal immune responses specific for the tumor, essentially creating a personalized vaccine. In this descriptive retrospective case report, those outcomes were not investigated and not part of this study. Research continues on developing more effective immune stimulators that further enhance immune responses and some of these are being investigated as immune therapies in cancer – often combined with other cancer immunotherapies. 8–10 Immune stimulant formulations that are used as adjuvants - like the one presented here - have been used for IT injection in human cancers and several have yielded promising results in human trials. These studies not only confirmed the immune mechanisms of action but showed efficacy against several chemotherapy-resistant cancers in human patients with advanced disease. Notably, the trial by Bahtia et al reported durable complete and partial multi-year remissions in human patients with local and metastatic Merkel cell carcinoma treated with IT injections of G100, a synthetic TLR4 agonist-based immune stimulator. 11 This was the first clinical trial of its kind and has garnered attention because of the remarkable results. 7,11 A phase1/2 randomized trial in human follicular lymphoma assessed clinical and immunologic outcomes in patients receiving Glucopyranosyl Lipid Adjuvant in Stable Emulsion (GLA-SE, aka G100) delivered IT (20 µg) following immune priming with low-dose radiation with or without a checkpoint inhibitor. The authors reported a 33% local response rate with 72% of 18 treated patients exhibiting abscopal tumor regression. 12 Seo et al described clinical and immunological outcomes in 12 human STS patients with metastatic disease and at least one superficial lesion accessible for IT injection, which received weekly IT doses of GLA-SE and radiation. After 8 doses in the 12 patients, divided into two cohorts (5 µg and 10 µg), local control was seen in all patients with one resulting in complete remission – a dramatic finding in a very difficult to treat cancer. 13 Sequencing the TCR β chain variable region from intratumoral T-cells identified several clonotypes that could also be found in peripheral circulation supporting the potential for a systemic abscopal effect, with only localized IT GLA-SE treatment. 13,14 Taken together, these reports support the conclusion that localized IT inflammation secondary to TLR4 agonist injection can generate systemic anticancer immunity. They also confirmed relatively low toxicity associated with administration of some immune stimulatory agents. 1 EmT4™ is our next generation TLR4 agonist-containing squalene emulsion adjuvant which contains the potent TLR4 activator 3D (6-acyl)-PHAD. This makes EmT4™ a functional mimic to GLA-SE, G100, and AS02. The agonist 3D (6-acyl)-PHAD is a synthetic mimic of the active congener found in naturally derived MPLA which has been used in approved human vaccines in the form of adjuvants AS01 and AS04 from GSK Bio plc (London, United Kingdom). EmT4™ has been extensively studied in murine models that detail its synthesis and validation of significant improvement in the strength and breadth of the immune response when used as a booster for mRNA vaccines such as with EmT4™-adjuvanted SARS-CoV-2 spike protein in mice. 15 There were no significant safety or health abnormalities noted in mice receiving EmT4™ with immunogen for the duration of the experiment. Research dogs receiving MPLA as a one-time treatment of 100 µg/kg did not display evidence of toxicity or abnormalities of concern in hematological and biochemical assays, predicting a similar EmT4™ safety profile in the dog. 5 A study by Larsen et al evaluated EmT4™ tolerability in several murine cohorts using FDA-like requirements monitoring daily weight and overt signs of morbidity including ruffled fur, lack of movement, and huddled or hunched posture. At the end of the 12-day study plasma ALT, AST liver enzyme levels and C-reactive protein (CRP) were evaluated in mouse-specific commercial ELISAs. Whole blood was analyzed for white blood cell, red blood cell, and platelet counts. There were no major differences in weight, liver enzyme concentrations and CRP levels between cohorts including the group that received 15 µg of EmT4™ on 4 consecutive days, totaling 60 µg although there was a small but significant elevation in total blood cell count in that group. There was no difference in organ weights compared to mice given EmT4™ once per week × 3 weeks. The authors concluded that when given as a single agent, EmT4™ was well-tolerated. 4 This immune stimulant is a highly effective trigger of humoral and cellular immune responses when given to mice intramuscularly with an antigen. EmT4™ was shown to activate mouse-derived macrophages and dendritic cells ex vivo . More recently, it has been investigated as an effective adjuvant in a Mycobacterium tuberculosis vaccine and proved to be highly effective at generating protective immunity by eliciting polyclonal CD4 Th1 T cell responses. 4 Our goal was to investigate the potential of utilizing EmT4™ as treatment for a tumor in a pet dog and potentially spare the animal from an invasive surgery in a location where surgical closure could be difficult. The extensive experience with MPLA by the authors provided highly informed expertise in this effort. This is a retrospective study which accounts for the absence of a biopsy prior to treatment in a slow growing tumor and given that the aspiration results did not confirm malignancy. It was never intended as a research study per se , however, the striking results compelled us to want to share these findings with the community as evidence of a promising, affordable immunotherapy that could potentially expand innovative veterinary cancer treatment options. The results of our study have generated interest in prospective controlled studies administering EmT4™ directly into canine tumors such as carcinomas, soft tissue sarcomas, melanomas, osseous sarcomas ( e.g ., osteosarcoma), and possibly others. The long-term goal is to explore developing clinical EmT4™ protocols that – perhaps combined with other therapeutic modalities ( e.g ., checkpoint inhibitors, low-dose radiation, certain chemotherapeutics) – will provide effective and durable anticancer protection mediated by the immune system in dogs, locally and systemically. To our knowledge, this is the first report of a canine receiving intratumoral injections of a TLR4 agonist containing adjuvant EmT4™ for a naturally occurring malignancy. Though EmT4™ is an MPLA mimetic - as are some other adjuvants - it differs because it is a defined synthetic molecule and therefore should have fewer off-target effects than natural mixtures of agonists. GSK’s approved commercial product 3D-MPL is derived by hydrolyzing LPS from Salmonella minnesota leading to the hydrolysis of any of the seven attached acyl chains and leaving a mixture of predominantly 4, 5, 6, and 7 acyl forms of the molecule. The one known to be active in humans is the hexaacyl form. The agonist in EmT4™ is a synthetic form of this hexaacyl structure and has therefore effectively been in millions of people receiving vaccinations. Case Patient An 8-yr-old female spayed Boston terrier (Fig. 2 A) belonging to one of the authors and who gave consent for treatment, was seen for a broad-based 3-cm soft tissue mass over the lateral side of the right elbow (Fig. 2 B). The lesion was non-painful and had been slowly enlarging for approximately 5 months. The mass was soft and fluctuant. During the observation period, it was aspirated for cytological evaluation on two separate occasions, yielding only adipose tissue each time. The mass was initially considered to be a lipoma. Due to its large size and continued slow progression, the owner requested mass excision, electing to have the tumor injected prior to surgery with the adjuvant EmT4™, based on extensive personal scientific knowledge of TLR4 agonists and expertise and after conferring with other scientists and MDs. The findings by Baldrick et al that showed a single high dose of MPLA in research dogs did not cause toxicity further underpinned this decision. 5 It was given IT two times separated by two weeks. The planned dose of EmT4™ was based on data from related TLR4 agonists. EmT4™ is similar to G100, which has been evaluated in murine models 16 and administered intratumorally (IT) repeatedly to human patients with follicular lymphoma with little to no toxicity. 12 Dogs receiving MPLA, a TLR4 agonist closely related to EmT4™, tolerated the injection well, i.e. , without clinical adverse events and normal hematologic and biochemical screens, when given as a single-dose of 100 µg/kg, although repeated daily treatments caused signs of toxicity, further discussed in Materials and Methods ( EmT4™ dose and injection schedule) . 5 The tumor grew after the first injection, from ~ 3 cm to 5 cm diameter. The skin became taught over the tumor and was sensitive to touch and warm. Maximum growth occurred about 3–6 hours after the injection. The dog experienced mild discomfort and lethargy, but was self-supporting, alert, responsive and ate and drank normally. These signs were compatible with a grade 1 Constitutional Signs adverse event according to the VCOG-CTCAE v.2. 17 We suspect that there was a spike in TNF-α within the tumor, and possibly systemically secondary to TLR4 activation. TNF-α is pyrogenic and could have induced fever although in this case body temperature wasn’t recorded at home. Fever would be consistent with the observation by Shi et al that reported transient spikes of TNF-α in the blood correlated with the time of fever several hours after administration of the macrophage activator L-MTP-PE intravenously to dogs with osteosarcoma. 18 L-MTP-PE is also a TLR4 agonist. 19 Within 24 hours, the tumor decreased in size but remained enlarged although no longer painful. After the second injection two weeks later, the tumor did not enlarge appreciably, and the dog did not show signs of discomfort. The tumor underwent an excisional biopsy four weeks after the second IT injection, when size measurements were relatively unchanged. She recovered from surgery uneventfully. The tumor was fixed in formalin and embedded in paraffin wax for sectioning. The diagnosis was STS, grade 2. The margins showed tumor cells extending to the cut edge of the tumor indicating residual tumor remained at the site. The margins were later treated with two rounds of electrochemotherapy. There has not been recurrence to date, approximately 28 months following EmT4™ treatment. Materials and Methods EmT4™ EmT4™ ( Emulsion with a Toll-Like Receptor 4 ligand ) is an MPLA analog molecule in a squalene emulsion. EmT4™ is produced by PAI Life Sciences, Inc (Seattle, WA) who provided it for this study. EmT4™ was prepared by incorporating synthetic 3D (6-acyl)-PHAD into a squalene-based stable oil-in-water emulsion using established methods. 4,15 The emulsion contained squalene, glycerol, and polysorbate 80 as stabilizers, with a particle size around 100 +/- 40 nm as measured by dynamic light scattering. The final formulation was sterile filtered, aseptically filled into 2 mL vials, and stored at 2–8°C until use. It was produced in an ISO7 GMP cleanroom following GLP protocols. It was tested post-production for sterility and particle size. EmT4™ dose and injection schedule Two IT injections of EmT4™ were given 2 weeks apart. The dose was deposited in several locations within the tumor by redirecting the needle without removing it. There were two separate IT injection sites for each treatment. The planned dose of EmT4™ was based on data from related TLR4 agonists. EmT4™ is similar to G100, which has been evaluated in murine models 16 and administered IT to human patients with follicular lymphoma at doses up to 40 µg weekly for eight weeks; adverse events were predominantly grade 1–2. 12 As mentioned above, MPLA - a compound closely related to EmT4™ - was well tolerated in dogs at a single dose of 100 µg/kg indicating a wide safety margin for single-dose administration, although repeated daily dosing led to immune overstimulation and toxicity. 5 These data supported the conclusion that doses well below 100 µg per dog are likely to be safe. Following intratumoral injection, the amount of EmT4™ retained within the tumor was estimated to be approximately 40 µg in a volume of ~ 0.2 mL, well below doses associated with toxicity in dogs. Histopathology Formalin-fixed paraffin-embedded tumor tissue obtained via excisional biopsy was sectioned at 4 microns, stained with hematoxylin and eosin, and evaluated by two ACVP board certified pathologists independently at two different institutions, Louisiana State University and IDEXX Laboratories. Immunohistochemistry Immunohistochemistry was performed for canine immune cell markers. The antibody clones used in this study have been previously utilized in published canine studies; specific references for each supports their use here. All IHC antibody markers have been tested in normal canine lymphoid tissue controls to confirm appropriate compartmental labeling. These include T cells (CD3, clone F7.2.38, Dako diluted 1:100) 20 , B cells (CD20, cat. #RB-9013-P, Thermo Scientific diluted 1:400) 21 , regulatory T cells (FoxP3, clone FJK-16S, Thermo Fisher diluted 1:100) 22 , macrophages, dendritic cells (Iba-1, FUJIFILM Wako Chemicals USA) 23 , and macrophages, histiocytes (CD204, clone SRA-E5, Abnova, diluted 1:200) 24 . Staining was done using the automated Leica BOND-MAX stainer according to published protocols. 25,26 Prior to staining, heat-induced epitope retrieval was performed with a citrate-based pH 6.0 solution (Leica Biosystems) for 20 min at 100 C. Diaminobenzidine was used as the chromogen, positive stained cells appearing brown. Slides were scanned on the PhenoImager HT 2.0 (Akoya Biosciences) and QuPath software v0.6.0 was used for qualitative and quantitative analysis of positive staining cells. RNAscope™ In Situ Hybridization RNAscope™ in situ hybridization (ISH) was used to detect RNA transcripts for CD4 , CD8A , IFNG , TNFA , and IL-2 within immune cells using FFPE tissue sections. The RNAscope™ ISH assay (Advanced Cell Diagnostics-ACD, Newark, CA) was performed using the RNAscope™ 2.5 LSx RED Reagent Kit on the automated BOND RXm platform (Leica Biosystems, Buffalo Grove, IL) as described previously. 26,27 Four-micron sections of FFPE tissues were mounted on positively charged Superfrost® Plus Slides (VWR, Radnor, PA, USA) and subjected to automated baking and deparaffinization, followed by heat-induced epitope retrieval using a ready-to-use EDTA-based solution (pH 9.0; Leica Biosystems) at 100° C for 15 min. Subsequently, tissue sections were treated with a ready-to-use protease (RNAscope™ 2.5 LSx Protease, ACD) for 15 min at 40° C, followed by a ready-to-use hydrogen peroxide solution for 10 min at room temperature. Slides were then incubated with the ready-to-use probe mixture for 2 h at 40° C, and the signal was amplified using a specific set of amplifiers (AMP1 through AMP6 as recommended by the manufacturer). The signal was detected using a Fast Red solution for 10 min at room temperature. Finally, slides were counterstained with a ready-to-use hematoxylin stain for 5 min, followed by five washes with 1× BOND Wash Solution (Leica Biosystems). Slides were rinsed in deionized water, dried in a 60° C oven for 30 min and mounted with Ecomount® (Biocare, Concord, CA, USA). Proprietary antisense probes were designed and provided by ACD using their algorithm for canine CD4 , CD8A , TNFA , IFNG , and IL-2 (ACD catalog # 459558, 459548, 578948, 830438, and 1830858 for an IL-2 custom designed probe, respectively). Probe sequences from ACD are proprietary and not provided to users. Specific information on the probes including GenBank accession number, number of ZZ pairs designed, and target region of the mRNA are provided on the company’s website. On-target binding specificity of probes was verified using normal canine lymphoid tissue, i.e. , lymph node, spleen, and tonsil as ISH controls. Slides were scanned on the PhenoImager HT 2.0 (Akoya Biosciences) and QuPath V0.6.0 was used for quantitative analysis of positive staining cells visualized as punctate red dots. 28 Results Histopathology The tumor was diagnosed as an STS, grade 2. Histopathologic examination revealed expansion of the subcutis by an unencapsulated, densely cellular and expansile neoplasm composed of neoplastic spindle cells arranged in streams and whorls and supported by a scant stroma (Fig. 2 C inset). Neoplastic cells exhibited moderate atypia and there were 14 atypical mitotic figures counted in 2.37mm 2 (equivalent to 10 high power fields). Intratumoral necrosis was not present in any of the examined sections. Together, the combined differentiation, mitotic, and tumor necrosis scores yielded a histologic total grade score of 4–5, considered intermediate grade/grade 2. 29 Within multiple regions of the neoplasm, neoplastic cells were more widely separated by intratumoral interstitial edema, and within a given region, prominent lymphocytic cuffs delimit approximately 50% of blood vessels with sporadic infiltrating lymphocytes within other areas of the neoplasm (Fig. 2 C, 2 D). There was no pre-EmT4™ injection sample available as multiple aspirates were non-diagnostic, and an interventional biopsy was not done given the slow growth of the tumor. Two archived FFPE tissue blocks at Louisiana Animal Disease Diagnostic Laboratory, Louisiana State University from two dogs with STS (right rear limb near the stifle and right forelimb lateral to the shoulder) of comparable histologies, grade, and cellular features are presented as examples of non-injected tumors in lieu of a pre-injected sample. However, these should not be construed as controls. Immunohistochemistry Immunohistochemistry for immune cell markers was performed on a selected FFPE tissue block representative of the treated STS, including CD3, CD20, FoxP3, CD204, and Iba-1. Four representative perivascular regions with large numbers of mononuclear cells were assessed and the proportion of cells immunolabeled by each specific IHC marker was determined. Within the cellular perivascular cuffs, CD20 + B lymphocytes represented the most frequent cellular population, followed by CD3 + T lymphocytes, Iba-1 + macrophages/dendritic cells, CD204 + macrophages/histiocytes, and FoxP3 + regulatory T cells (Figs. 3 , 4 ). LPS can activate B cells through paracrine signals as well as through B cell receptor binding of the associate carbohydrates – thereby inducing cell proliferation and differentiation. 30 This may help to explain why there were large numbers of B cells in the clusters as EmT4™ activates the same signaling cascade as LPS and should have similar effects on these cells. Abscopal effects are usually attributed to T cell-mediated immunity, but B cells also contribute through antibody production and cytokine signaling that modulate T cell activity. The presence of tertiary lymphoid structures rich in activated B cells correlates with better systemic and abscopal immune responses. 31 The mean number of cells in four fields positive for T cells (CD3), regulatory T cells (FoxP3), B cells (CD20), macrophages/histiocytes (CD204), and macrophages/dendritic cells (Iba-1) are expressed as percentage of the total mononuclear cell population scanned (Table 1 ), as described. 25 Sporadic CD3 + T lymphocytes and FoxP3 + regulatory T lymphocytes were detected elsewhere within the neoplasm, while no CD20 + B lymphocytes were identified beyond the cell clusters. Infiltration by CD204 positive histiocytes is widespread throughout the interstitium of the neoplasm, considered to be resident cells and not due to EmT4™ treatment. 32 Table 1 Percent Positive in Immune Cell Clusters (mean) Boston Terrier FS 8yr (patient) CD3 FoxP3 CD20 CD204 Iba-1 24.6 SD 7.21 8 SD 1.48 57.2 SD 7.21 10.2 SD 4.55 36.2 SD 11.36 Catahoula Hound MC 8yr (archived tissue) 0.00 0.00 0.00 18.53 SD 10.67 11.44 SD 3.93 Mixed Breed FS 11yr (archived tissue) 0.00 0.00 0.00 10.32 SD 15.83 9.46 SD 4.03 IHC slides were digitized and analyzed with digital pathology software (QuPath for positive cell detection and quantitative analysis). Four representative perivascular regions with large numbers of mononuclear cells were assessed for signal generated by each immune marker. The mean number of cells positive for T cells (CD3), regulatory T cells (FoxP3), B cells (CD20), and macrophages/dendritic/histiocytic cells (CD204, Iba-1) are expressed as percentage of the total mononuclear cell population scanned. In the two archived tumors that did not receive EmT4™ injection, there were no cells positive for CD3, FoxP3, and CD20 lymphocytes (Table 1 , Fig. 4 ). CD204 + macrophages were similarly abundant and with a similar distribution in the injected and non-injected tumors. The marker Iba-1 that identifies macrophages and dendritic cells, differentiated between the injected tumor and the two non-injected tumors having approximately three times as many Iba-1 positive cells (36%) within perivascular regions compared to 9% and 11% for the non-injected tumors. RNAscope In Situ Hybridization (ISH) Four separate densely cellular, perivascular fields were examined for ISH signals and presented as the mean % positive cells (+/- SD) of the total cell count in the 4 fields analyzed (Table 2 ). Table 2 Percent Cells Positive by ISH (mean) CD4 CD8 TNF-α IFN-g IL-2 29.00% SD 6.18 3.20% SD 1.09 23.55% SD 6.43 1.31% SD 0.8 0.12% SD 0.084 In situ hybridization revealed that the highest proportion of positive transcripts were for CD4 and TNFA at approximately 30% and 24%, respectively (Table 2 , Fig. 5 ). Although present, CD8A -positive cells were appreciably less than CD4 , and IFNG was detected at a low level. IL2 -positive cells were low in numbers. Discussion and Conclusions This case study investigated tolerability and local immune responses following IT injection of a unique TLR4 agonist formulation in a canine STS. It is a case report that includes two archived non-injected canine tumors of comparable histology. Two independent attempts by the attending veterinarian to obtain a diagnostic cytologic sample by aspiration yielded only non-diagnostic adipose tissue - which is not uncommon for sarcomas that don’t always exfoliate when aspirated. For this reason, the mass was initially presumed to be a lipoma and surgical removal was considered an elective procedure at that time and not immediately done. The non-injected dog tumors with similar STS histologic features and grade as the treated tumor were utilized in lieu of the absence of a pre-injection sample. They served for comparison with post IT EmT4™ but not as controls. Though not ideal, this was helpful to illustrate stark differences. Generally, canine STS do not exhibit pronounced immune responses due to the presumed low mutational burden resulting in fewer tumor antigens. 33 High grade, poorly differentiated canine STS on the other hand, likely have higher mutational burdens, and have been reported to be infiltrated with CD3, CD20, and FoxP3 lymphocytes to varying degrees. Myxosarcomas and perivascular wall tumors appear to be the most antigenic. 34 The STS from the dog of this report is considered intermediate grade based on moderate cell atypia and intermediate mitotic fraction, 14 atypical mitotic figures/2.37mm 2 (equivalent to 10 high power fields), without necrosis. 29 The archived STS from two dogs did not contain any lymphocytes within the tumor parenchyma. Avallone et al reported that the canine fibrosarcomas examined in their study had no or low numbers of lymphocytes with counts statistically below those of myxosarcomas and perivascular wall tumors. 34 Without a pre-injection tissue sample to serve as a control, we cannot know what the cellular landscape was or even if there were already immune cell infiltrates present. After the first EmT4™ treatment but not the second, the dog was less active and preferred to lay down but alert and responsive the evening of the injection. After the first injection, but not the second, the tumor swelled rapidly and the overlying skin became taught, warm, and painful on touch, going from 3 to 5 cm. This is a classic feature of pseudoprogression, a hallmark of immune therapy, and was not considered tumor hyperprogression, since the tumor rapidly partially regressed in size a few days after swelling. 35 Activation of TLR4 receptors expressed by dendritic cells and macrophages within the tumor by EmT4™ would result in release of proinflammatory cytokines like TNF-α and chemokines. These would lead to the influx of immune cells and rapid swelling of the tumor due to edema and the volume of cells now occupying the sarcoma. The release of cytokines was confirmed by RNA hybridization with a TNF-α probe showing transcripts in 23.6% of clustered immune cells. TLR4 signaling is tightly regulated to prevent excessive inflammation and autoimmunity. 36 Consistent with clinical experience for many immunotherapies, adverse events are most prominent after initial dosing and are often milder or absent with subsequent administrations - including for TLR4 agonists due to autoregulation of strong initial innate responses. 36,37 In this study, the second dose of EmT4™ did not elicit the adverse events observed after the first dose, yet remained immunostimulatory, as evidenced by TNF-α transcripts detected in immune cells four weeks after the second injection. Given the short half-life of TNF-α protein and transcripts in both humans and mice, 38 these findings indicate sustained immune activation up to the time of tumor excision. This pattern is consistent with a TLR4 regulatory model in which the first dose delivers a “danger signal and reset,” while the second dose provides immune instruction without triggering overt inflammation, potentially explaining the transient tumor enlargement observed after the initial treatment. Although there was not a pre-injection sample for valid comparison, it is reasonable to conclude that these signs were associated with EmT4™ due to the rapid response after injection of the formulation and compatible with acute inflammation that was not present pre-injection. Further, TNF-α transcripts were found in the RNA hybridization assay implying its continual synthesis 4-weeks after the last EmT4™ injection. The histologic features of this tumor — absence of immune cells in the two non-injected, histologically comparable STS, presence of T cells, B cells, and macrophages within the injected tumor milieu forming dense perivascular cell aggregates — taken together may suggest EmT4™ IT induced a local inflammatory immune response. The infiltrates in the TME track closely with what has been described for murine and human tumors injected with related LPS mimetics or immunostimulatory compounds. Perivascular cuffing by immune cells is a feature of some infectious diseases ( e.g. , canine distemper, rabies, etc). In cancer it is considered an immune response to the tumor. 39 Using murine models of human colorectal and pancreatic cancer, Stoltzfus et al employed multimodal spatial analysis of immune cells clustered around intratumoral vasculature to investigate microanatomical organization of cellular infiltrates in unperturbed tumors and following immunotherapy. While perivascular immune cells were present in untreated tumors, following immunotherapy they were markedly increased. 39 The study also revealed that T cells co-localized with dendritic cells or activated macrophages in compact bundles. In our study, IHC revealed co-mingling of T and B cells with macrophages within the clusters (Fig. 3 ). This may have been due to a direct effect of EmT4™; a pretreatment biopsy would have been conclusive that this was indeed the case. The finding of TNF-α in immune cells using RNA hybridization supports the conclusion that the macrophages within clusters were activated. Stoltzfus et al also reported that cell clusters were not found around all vessels, as was also the case in our observations where approximately 50% of tumor vasculature showed perivascular immune cell cuffs. 39 This raises interesting questions as to what explains the differences. Could there have been heterogeneity in the vasculature associated with cell clusters versus vessels not associated with cell clusters? Neovasculature within tumors can be disorganized and it is possible that there were differences in adhesion molecules expressed by endothelial cells that dictated which vessels would serve as entry points of immune cells into the tumor. This remains speculative and has yet to be determined. Generally, immune cells in the injected STS remained in thick perivascular clusters without dissemination into the deeper tumor. This may be due to where the EmT4™ was deposited: Given the tumor’s large size and the small needle used for injection, EmT4™ may not have been deposited deeply into the tumor parenchyma. In the two rodent models described by Stoltzfus et al immune cells were not found in the deeper tumor tissue either. 39 Those tumors were not subjected to IT administration of the immunostimulants that the mice received. The investigators suggested immunosuppressive factors ( e,g. , hypoxia and others) may have prevented migration of immune cells from peripheral perivascular nests to the interior and is a possible explanation for what we saw. The persistence of dense layers of immune cells in perivascular spaces may be considered perivascular immune nests, immune-rich microenvironments capable of mediating immune effects in situ . 39 The in situ RNA hybridization data provided insight into the function of the cell infiltrates. It suggests that EmT4™ may not only have attracted lymphocytes to the TME but also activated immune function genes to produce transcripts for the proinflammatory cytokine TNF-α in abundance. High levels of CD4 lymphocytes and TNF-α, as described here, are hallmarks of a Th1 biased immune response. Seo et al concluded that IT administration of the MPLA mimetic GLA-SE induced a Th1 response based on the predominance of CD4 lymphocyte infiltrates accompanied by relatively high concentrations of TNF-α within human STS. 13 Compared to TNF-α, IFN-g expression was low but nevertheless present in some cells in the post-EmT4™ sample. The same can be said about CD8 positive cells with only ~ 3% positivity. This may be due to the relatively short time interval ( i.e ., 4 weeks) between the second EmT4™ injection and tumor excision as immunotherapy often takes more than 8 weeks to induce pronounced cytotoxic effects. 40 It is therefore possible that had there been a longer observation period, there might have been a higher percentage of CD8 cells within the tumor. Notably, the human STS patients reported by Seo et al received a total of 8 IT injections of GLA-SE – one per week compared to 2 doses of EmT4™ given to the dog of this report. There were also large numbers of B cells present in the lymphocyte clusters. The report by Venkataraman et al confirmed LPS induces B cell proliferation and identified signaling pathways in B cells contributing to this response. 30 Expression of TLR4 in murine B cell lymphoma is important for the therapeutic activity of these agonists when given IT. 16 Additional studies are needed to confirm the direct effect EmT4™ may have had in recruiting large numbers of B cells to the TME following IT administration to the dog’s STS. However, LPS is a potent stimulator of B cell proliferation and a role for EmT4™ exerting a similar effect cannot be ruled out. The presence of B cells adds to the panoply of immune cells that appear to have trafficked into the TME in a coordinated response induced by the two IT EmT4™ injections. Only low numbers of FoxP3 regulatory T lymphocytes were present supporting an active, not suppressive, inflammatory process. Myeloid-derived suppressor cells were not examined and merit further attention in the future. Interestingly, Richert et al reported that a liposome formulation of a TLR4 agonist in their murine experimental osteosarcoma model could reverse the polarization of M2 macrophages shifting to an M1 phenotype, creating a proinflammatory TME. 19 Indeed, classically activated macrophages (M1-like) result from LPS or mimetic-triggered signaling through TLR4 and produce inflammatory cytokines that favor anti-tumor immune responses. 41 The ability of TLR4 agonists such as EmT4™ to switch macrophage polarity from immunosuppressive to immunostimulatory phenotypes, alone or in combination therapy, is potentially an added benefit of IT administration of TLR4 agonists that prompts further investigation. Additional dogs with STS (or other histologies) are needed with biopsies pre-EmT4 IT injection, to conclude that EmT4 not only promotes tumor infiltration by immune cells, but it also activates them within the tumor parenchyma. Tumor antigens that are released by immunogenic cell death, are scavenged and processed by macrophages and dendritic cells that present tumor epitopes to T cells. The activated T cells migrate and multiply in draining lymph nodes expanding anti-tumor clonotypes that can circulate (Fig. 1 ). These cells mediate MHC-restricted tumoricidal effects with specificity and can traffic back to the TME of the primary tumor as well as generating abscopal cytotoxicity at secondary tumor locations. Several publications have reported identical clones in the circulation and in the TME following IT administration of TLR4 agonists G100 and GLA-SE confirming this to be the case. 11,13 It will be interesting to determine if similar immunologic events occur following EmT4™ IT treatment in canine cancer. Expanded clinical studies with IT EmT4™ are needed to pursue a long-term goal of combining it with other regimens in dogs with cancer. While it is unlikely that EmT4™ would solely be used as a standalone therapy, it would be straightforward and immunologically sound to include it in combination with other established treatment strategies. As an example, low doses of radiation have an immunostimulatory effect, inducing immunogenic cell death releasing damaged-associated molecular patterns (DAMPs) that activate dendritic cells and macrophages as well as releasing tumor antigens and could be synergistic with the immune activation induced by EmT4™. 42 Human patients in Merkel cell carcinoma and follicular lymphoma trials received low dose radiation treatment prior to IT TLR4 treatments. 11,12 Higher doses of radiation prior to TLR4 administration were used by Seo et al treating high grade STS. 13 Treatment schedules often resemble those of palliative radiation, hypofractionation, which veterinary oncology has employed for many years and clients readily elect this form of treatment. 43,44 With further evidence, it would be convenient to add EmT4™ to these treatments as part of clinical trials to further improve outcomes. Combining TLR agonists with checkpoint inhibitors has proven to be an effective regimen in several human cancers. 45,46 With the advent of checkpoint inhibitors in veterinary oncology, IT EmT4™ combined with an anti-PD1 antibody, anti-LAG3 antibody, or anti-CTLA-4 antibody could offer new opportunities to potentiate responses. Combination immunotherapy has been shown to be more effective than single agent. 47 Additionally, certain chemotherapeutic agents can induce immunogenic cell death including anthracyclines, certain platinum drugs, cyclophosphamide and others. 48 Cyclophosphamide given at low doses induces immunogenic cell death and can reduce the numbers of regulatory T cells. 49–52 Combined with a TLR4 agonist such as IT EmT4™, the TME could be tipped in favor of Th1 biased anti-tumor immunity. Selective killing of regulatory T cells by low dose cyclophosphamide would enhance potent immune triggering by TLR4 activation. 50,52 Although in this study FoxP3 + cells comprised only 8% of the intra-tumoral lymphocytes, the CD3/FoxP3 ratio is approximately 3. Compared with radiotherapy or monoclonal antibody treatments, the chemotherapy approach could be a novel, inexpensive, immunotherapeutic option when combined with IT TLR4 activation. It would likely be accepted by dog owners because of its simplicity and could be provided at a lower cost compared to surgery. Activating TLR4 with EmT4™ could also potentially reduce the number of concurrent treatments with other costly immunotherapeutics by enhancing their efficacy when used in combinations. Keeping in mind this is a case report of one dog, more experience with EmT4™ is needed prospectively – including pre-surgical biopsy – to validate and extend the findings presented here. This single case study has provided valuable insight into a promising immune treatment that could potentially be scaled and find a place in the expanding veterinary immunotherapy quiver. Based on the dog in this report, EmT4™ appears to be nontoxic and was simple and quick to administer, minimizing restraint and stress for the dog. For these reasons - as well as its powerful immune-stimulatory properties - acceptance by dog owners could become a reality, but only after future studies confirm and expand on the findings presented here. Abbreviations TLR4 toll–like receptor 4 STS soft tissue sarcoma LPS lipopolysaccharide EmT4™ Emulsion with a Toll–Like Receptor ligand 4 MPLA monophosphoryl lipid A IFN g–interferon gamma TNF α–tumor necrosis factor alpha IL 2–interleukin–2 TME tumor microenvironment Declarations Ethics approval and consent to participate The dog owner provided consent and ethical considerations were discussed in detail by the dog owner and veterinarian, as well as with the other authors. Consent for publication Not applicable. Competing interests DC is the owner of PAI Life Sciences Inc. SG and JK are employees of PAI Life Sciences Inc., which holds intellectual property relevant to EmT4™, and produces EmT4™. Funding Canine Cancer Alliance PAI Life Sciences, Inc. Author Contribution SCH analyzed data and wrote the main manuscript with co-authors. SCH and MC prepared figures. MC performed the histologic, IHC, and ISH assays for the figures. JK helped with producing the tumor slices that were analyzed and assisted with developing the manuscript and interpreting the data. DRM provided veterinary care. SG and DC conceptualized the study. DC invented EmT4™, wrote the records used to produce the study article, and interpreted the data. All authors reviewed and discussed the data and edited the manuscript. Acknowledgements We thank the Canine Cancer Alliance for its generous support of this study. Data Availability All data generated or analyzed during this study are included in this published article. References Aznar MA, Tinari N, Rullán AJ, Sánchez-Paulete AR, Rodriguez-Ruiz ME, Melero I. Intratumoral delivery of immunotherapy-Act locally, think globally. J Immunol. 2017;198(1):31 − 9. doi: 10.4049/jimmunol.1601145. PMID: 27994166. Taleghani N, Bozorg A, Azimi A, Zamani H. Immunogenicity of HPV and HBV vaccines: adjuvanticity of synthetic analogs of monophosphoryl lipid A combined with aluminum hydroxide. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8982168","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Case Report","associatedPublications":[],"authors":[{"id":607747721,"identity":"ea145fb0-0e14-4946-88d9-ac07aef6922f","order_by":0,"name":"Stuart C Helfand","email":"data:image/png;base64,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","orcid":"","institution":"PAI Life Sciences, Inc","correspondingAuthor":true,"prefix":"","firstName":"Stuart","middleName":"C","lastName":"Helfand","suffix":""},{"id":607747726,"identity":"89b4ea7b-74d0-4c02-9ef5-d1ef80fa3fdd","order_by":1,"name":"Mariano Carossino","email":"","orcid":"","institution":"Louisiana State University","correspondingAuthor":false,"prefix":"","firstName":"Mariano","middleName":"","lastName":"Carossino","suffix":""},{"id":607747731,"identity":"6be4ecd0-f9cc-4455-a43d-5f5851eb9dfb","order_by":2,"name":"Jiho Kim","email":"","orcid":"","institution":"PAI Life Sciences, Inc","correspondingAuthor":false,"prefix":"","firstName":"Jiho","middleName":"","lastName":"Kim","suffix":""},{"id":607747734,"identity":"0b82e041-8fb4-42fa-b07b-e8bf9bd91848","order_by":3,"name":"David R McMonigle","email":"","orcid":"","institution":"McMonigle Veterinary Hospital","correspondingAuthor":false,"prefix":"","firstName":"David","middleName":"R","lastName":"McMonigle","suffix":""},{"id":607747737,"identity":"ca641f20-e26d-4a26-bc48-abe890551a0d","order_by":4,"name":"Darrick Carter","email":"","orcid":"","institution":"PAI Life Sciences, Inc","correspondingAuthor":false,"prefix":"","firstName":"Darrick","middleName":"","lastName":"Carter","suffix":""},{"id":607747739,"identity":"01a259c6-e9c2-479b-984a-248d9b991588","order_by":5,"name":"Sean A Gray","email":"","orcid":"","institution":"PAI Life Sciences, Inc","correspondingAuthor":false,"prefix":"","firstName":"Sean","middleName":"A","lastName":"Gray","suffix":""}],"badges":[],"createdAt":"2026-02-27 00:53:41","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8982168/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8982168/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104887810,"identity":"42b0eb15-2641-4c1b-bc1d-a87b96e33ab4","added_by":"auto","created_at":"2026-03-18 10:12:40","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":6467524,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eMechanism of action of TLR4 agonist antitumoral activity. \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e(1)\u003c/strong\u003e The agonist is injected into the tumor and \u003cstrong\u003e(2)\u003c/strong\u003e activates TLR4 expressed by dendritic cells and macrophages present within the tumor, \u003cstrong\u003e(3)\u003c/strong\u003e producing cytotoxic proinflammatory cytokines causing \u003cstrong\u003e(4)\u003c/strong\u003e immunogenic cell death \u003cstrong\u003e(5)\u003c/strong\u003e releasing tumor antigens that are recognized, phagocytosed, and processed by dendritic cells \u003cstrong\u003e(6)\u003c/strong\u003e further stimulated by the TLR4 agonist to mature, activate and \u003cstrong\u003e(7)\u003c/strong\u003e present tumor antigens to T cells in draining lymph nodes where \u003cstrong\u003e(8)\u003c/strong\u003e polyclonal T cells expand, circulate, and \u003cstrong\u003e(9)\u003c/strong\u003e mediate antigen-specific tumor cytotoxicity locally and systemically.\u003csup\u003e7\u003c/sup\u003e (Reprinted and adapted from Clinical Cancer Research, 2019; 25(4), 1127-1129, Marquez-Rodas I, et al, For Whom the Cell Tolls? Intratumoral Treatment Links Innate and Adaptive Immunity, with permission from AACR).\u003c/p\u003e","description":"","filename":"Figure1v2.png","url":"https://assets-eu.researchsquare.com/files/rs-8982168/v1/0cf49ba5be41ea06d0eece99.png"},{"id":104887767,"identity":"256f8687-4b90-41aa-bfdf-ca12533df7f3","added_by":"auto","created_at":"2026-03-18 10:12:38","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":33425444,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eSoft tissue sarcoma. \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e(A)\u003c/strong\u003eFemale spayed 8-year-old Boston Terrier given EmT4™ intratumorally. \u003cstrong\u003e(B)\u003c/strong\u003eSoft, 3 cm diameter, broad-based soft tissue sarcoma arising over the right elbow. \u003cstrong\u003e(C)\u003c/strong\u003e Low power image of hematoxylin and eosin-stained tissue section from the STS following excision four weeks after the second EmT4™ IT injection. Approximately 50% of blood vessels exhibited perivascular immune cell cuffing. Magnification 50X. A higher power (inset, 200X magnification) shows spindle cells in whorls and streams.\u003cstrong\u003e (D)\u003c/strong\u003e High power view of the large cell cluster in 2C. Dense, tightly packed accumulations of lymphocytes surround the vessel (black arrow). Magnification 200X.\u003c/p\u003e","description":"","filename":"Figure2v2.png","url":"https://assets-eu.researchsquare.com/files/rs-8982168/v1/5d62a9245e577acae96d0388.png"},{"id":104887907,"identity":"3e220e36-fc44-4e3b-9882-b62b84fa2253","added_by":"auto","created_at":"2026-03-18 10:12:56","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":30888157,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eImmunophenotypes of mononuclear cells within the EmT4™-injected STS.\u003c/strong\u003e\u003c/em\u003e Dense clusters of perivascular lymphocytes were stained for immune cell markers. Positive-staining cells for each marker appear brown. These aggregates were composed primarily of T cells (CD3, upper left) and B cells (CD20, lower left), with approximately twice as many B cells as T cells. Regulatory T cells (FoxP3, upper right) are present in small numbers and macrophages (CD204, lower right) comprised a small minority of the immune cell infiltrates. Non-neoplastic tissue histiocytes, widely scattered throughout the peritumoral parenchyma, also express CD204. Magnification 200×.\u003c/p\u003e","description":"","filename":"Figure3v2.png","url":"https://assets-eu.researchsquare.com/files/rs-8982168/v1/8e2a2b9d794ec91d62248ebc.png"},{"id":104888028,"identity":"39681a97-39df-45bc-a33c-e1f124d0e910","added_by":"auto","created_at":"2026-03-18 10:13:10","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":20646013,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eComparison of immune cell marker expression in EmT4™ injected STS (top row) and two non-injected STS (bottom two rows).\u003c/strong\u003e\u003c/em\u003e The untreated tumors were from archived biopsies of similar canine STS grade and cell morphology. They were both negative for expression of immune cell markers CD3, CD20, and FoxP3. Resident, non-neoplastic intratumoral histiocytes and dendritic cells are immunolabeled with antibodies specific for macrophages and histiocytes (CD204) and the macrophage/dendritic cell marker Iba-1, and widespread throughout the tumor parenchyma in all 3 tumors. Notably, Iba-1+ cells were present in greater numbers in the immune cell clusters compared to CD204+ cells (upper right two panels). There were fewer Iba-1+ histiocytes compared to CD204+ histiocytes in all 3 tumors. Black arrows point to vessels within each tumor. The gold ring around the vessel in the FoxP3 stained untreated tumor, second column bottom, is a processing artifact. Magnification 200X.\u003c/p\u003e","description":"","filename":"Figure4v2.png","url":"https://assets-eu.researchsquare.com/files/rs-8982168/v1/8a1d732f611f78356dc113a1.png"},{"id":104887896,"identity":"afa1ca28-e4b6-476d-bd4a-1ecfe2bc0390","added_by":"auto","created_at":"2026-03-18 10:12:53","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":6019957,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eIn situ\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ehybridization. \u003c/strong\u003e\u003cem\u003eIn situ\u003c/em\u003ehybridization using RNAscope™, was employed to detect CD4 (left) and TNF-a (right) transcripts in EmT4™ injected STS immune cell infiltrates. These probes yielded the brightest signal of the five different probes examined. Signal was detected with Fast Red and positive hybridization appears as punctate red dots, quantitated with imaging software.\u003c/p\u003e","description":"","filename":"Figure5v2.png","url":"https://assets-eu.researchsquare.com/files/rs-8982168/v1/96d2bf4a511af2edf6cd0f37.png"},{"id":105034552,"identity":"dc227633-bb46-46ea-855f-dfcbf6b283de","added_by":"auto","created_at":"2026-03-20 07:23:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":87793626,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8982168/v1/1d328d9d-344a-47a0-8c19-51cd7b435c9f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Intratumoral injection of a novel TLR4 agonist elicits infiltration by immune effector cells in a canine soft tissue sarcoma: An Immunologic Case Study","fulltext":[{"header":"Background","content":"\u003cp\u003eA major goal of cancer immunotherapy is to modify the tumor microenvironment (TME) so that conditions favor a tumor-specific immune response. Multiple mechanisms within the TME prevent immune effector cells from attacking the cancer even if the cells manage to penetrate the tumor parenchyma. Some tumors are devoid of effector cells because they have a low mutational burden and/or express fewer tumor antigens. The lack of tumor neoantigens can reduce influx of immune effector cells into the TME, thereby preventing a tumor-specific immune response. Once activated, the immune system can be highly effective against cancer as demonstrated by remarkable results observed when using check point inhibitors and other therapeutic monoclonal antibodies. One strategy to reprogram the TME is by directly injecting immune stimulants within the tumor architecture to induce immunologic hotspots \u003cem\u003ein situ\u003c/em\u003e.\u003csup\u003e1\u003c/sup\u003e In this report, intratumoral (IT) immune infiltration is described in a canine soft tissue sarcoma (STS) following intralesional administration of an innate immune system activator.\u003c/p\u003e \u003cp\u003eWe used the adjuvant EmT4\u0026trade; which contains a synthetic mimetic of monophosphoryl lipid A (MPLA) called 3D (6-acyl)-PHAD, the core structure of bacterial lipopolysaccharide (LPS). It targets and activates Toll-like receptor 4 (TLR4) without the toxicity associated with LPS.\u003csup\u003e2\u003c/sup\u003e Triggering TLR4 and its signaling pathways can lead to potent innate immune responses. TLR4 is evolutionarily conserved across mammalian species indicating its importance in immune responses - especially in response to gram negative bacterial LPS. For example, \u003cem\u003eLeptospira\u003c/em\u003e vaccines for dogs trigger identical immune responses in multiple species through TLR activation. Canine monocyte-derived dendritic cells were used in the study to verify canine expression and activation by MPLA.\u003csup\u003e3\u003c/sup\u003e Since MPLA activates TLR4 in the dog and the active ingredient of EmT4\u0026trade; is a synthetic mimetic of MPLA, we hypothesized that canine immune cells would efficiently respond to injection of this immune stimulant.\u003csup\u003e4,5\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThis activator is modeled on adjuvants used in vaccines that turn on robust immune responses by binding pattern recognition receptors or TLRs on dendritic cells and macrophages. Activation of the TLR4 signaling pathway results in induction of pro-inflammatory genes - including IL-1β, TNF-α, and type 1 interferon \u003cb\u003e-\u003c/b\u003e which in turn upregulate TLR4 expression and immune cell recruitment. Chemokines and cytokines induced by TLR4 activation can mediate immunogenic tumor cell death. Tumor antigens phagocytosed and processed by activated dendritic cells and macrophages are presented to T-cells within the tumor and local draining lymph nodes.\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eT cells that traffic to draining lymph nodes can expand and are then capable of returning to the tumor \u003cem\u003een masse\u003c/em\u003e as well as circulating systemically and mediating abscopal cytotoxicity at metastatic sites (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003csup\u003e7\u003c/sup\u003e The steps in this process recapitulate those following vaccination with adjuvants for infectious diseases and can be considered \u003cem\u003ein situ\u003c/em\u003e autovaccination against a cancer that facilitates polyclonal immune responses specific for the tumor, essentially creating a personalized vaccine. In this descriptive retrospective case report, those outcomes were not investigated and not part of this study. Research continues on developing more effective immune stimulators that further enhance immune responses and some of these are being investigated as immune therapies in cancer \u0026ndash; often combined with other cancer immunotherapies.\u003csup\u003e8\u0026ndash;10\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eImmune stimulant formulations that are used as adjuvants - like the one presented here - have been used for IT injection in human cancers and several have yielded promising results in human trials. These studies not only confirmed the immune mechanisms of action but showed efficacy against several chemotherapy-resistant cancers in human patients with advanced disease. Notably, the trial by Bahtia \u003cem\u003eet al\u003c/em\u003e reported durable complete and partial multi-year remissions in human patients with local and metastatic Merkel cell carcinoma treated with IT injections of G100, a synthetic TLR4 agonist-based immune stimulator.\u003csup\u003e11\u003c/sup\u003e This was the first clinical trial of its kind and has garnered attention because of the remarkable results.\u003csup\u003e7,11\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eA phase1/2 randomized trial in human follicular lymphoma assessed clinical and immunologic outcomes in patients receiving Glucopyranosyl Lipid Adjuvant in Stable Emulsion (GLA-SE, aka G100) delivered IT (20 \u0026micro;g) following immune priming with low-dose radiation with or without a checkpoint inhibitor. The authors reported a 33% local response rate with 72% of 18 treated patients exhibiting abscopal tumor regression.\u003csup\u003e12\u003c/sup\u003e Seo \u003cem\u003eet al\u003c/em\u003e described clinical and immunological outcomes in 12 human STS patients with metastatic disease and at least one superficial lesion accessible for IT injection, which received weekly IT doses of GLA-SE and radiation. After 8 doses in the 12 patients, divided into two cohorts (5 \u0026micro;g and 10 \u0026micro;g), local control was seen in all patients with one resulting in complete remission \u0026ndash; a dramatic finding in a very difficult to treat cancer.\u003csup\u003e13\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eSequencing the TCR β chain variable region from intratumoral T-cells identified several clonotypes that could also be found in peripheral circulation supporting the potential for a systemic abscopal effect, with only localized IT GLA-SE treatment.\u003csup\u003e13,14\u003c/sup\u003e Taken together, these reports support the conclusion that localized IT inflammation secondary to TLR4 agonist injection can generate systemic anticancer immunity. They also confirmed relatively low toxicity associated with administration of some immune stimulatory agents.\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eEmT4\u0026trade; is our next generation TLR4 agonist-containing squalene emulsion adjuvant which contains the potent TLR4 activator 3D (6-acyl)-PHAD. This makes EmT4\u0026trade; a functional mimic to GLA-SE, G100, and AS02. The agonist 3D (6-acyl)-PHAD is a synthetic mimic of the active congener found in naturally derived MPLA which has been used in approved human vaccines in the form of adjuvants AS01 and AS04 from GSK Bio plc (London, United Kingdom). EmT4\u0026trade; has been extensively studied in murine models that detail its synthesis and validation of significant improvement in the strength and breadth of the immune response when used as a booster for mRNA vaccines such as with EmT4\u0026trade;-adjuvanted SARS-CoV-2 spike protein in mice.\u003csup\u003e15\u003c/sup\u003e There were no significant safety or health abnormalities noted in mice receiving EmT4\u0026trade; with immunogen for the duration of the experiment. Research dogs receiving MPLA as a one-time treatment of 100 \u0026micro;g/kg did not display evidence of toxicity or abnormalities of concern in hematological and biochemical assays, predicting a similar EmT4\u0026trade; safety profile in the dog.\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eA study by Larsen \u003cem\u003eet al\u003c/em\u003e evaluated EmT4\u0026trade; tolerability in several murine cohorts using FDA-like requirements monitoring daily weight and overt signs of morbidity including ruffled fur, lack of movement, and huddled or hunched posture. At the end of the 12-day study plasma ALT, AST liver enzyme levels and C-reactive protein (CRP) were evaluated in mouse-specific commercial ELISAs. Whole blood was analyzed for white blood cell, red blood cell, and platelet counts. There were no major differences in weight, liver enzyme concentrations and CRP levels between cohorts including the group that received 15 \u0026micro;g of EmT4\u0026trade; on 4 consecutive days, totaling 60 \u0026micro;g although there was a small but significant elevation in total blood cell count in that group. There was no difference in organ weights compared to mice given EmT4\u0026trade; once per week \u0026times; 3 weeks. The authors concluded that when given as a single agent, EmT4\u0026trade; was well-tolerated.\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThis immune stimulant is a highly effective trigger of humoral and cellular immune responses when given to mice intramuscularly with an antigen. EmT4\u0026trade; was shown to activate mouse-derived macrophages and dendritic cells \u003cem\u003eex vivo\u003c/em\u003e. More recently, it has been investigated as an effective adjuvant in a \u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e vaccine and proved to be highly effective at generating protective immunity by eliciting polyclonal CD4 Th1 T cell responses.\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eOur goal was to investigate the potential of utilizing EmT4\u0026trade; as treatment for a tumor in a pet dog and potentially spare the animal from an invasive surgery in a location where surgical closure could be difficult. The extensive experience with MPLA by the authors provided highly informed expertise in this effort. This is a retrospective study which accounts for the absence of a biopsy prior to treatment in a slow growing tumor and given that the aspiration results did not confirm malignancy. It was never intended as a research study \u003cem\u003eper se\u003c/em\u003e, however, the striking results compelled us to want to share these findings with the community as evidence of a promising, affordable immunotherapy that could potentially expand innovative veterinary cancer treatment options.\u003c/p\u003e \u003cp\u003eThe results of our study have generated interest in prospective controlled studies administering EmT4\u0026trade; directly into canine tumors such as carcinomas, soft tissue sarcomas, melanomas, osseous sarcomas (\u003cem\u003ee.g\u003c/em\u003e., osteosarcoma), and possibly others. The long-term goal is to explore developing clinical EmT4\u0026trade; protocols that \u0026ndash; perhaps combined with other therapeutic modalities (\u003cem\u003ee.g\u003c/em\u003e., checkpoint inhibitors, low-dose radiation, certain chemotherapeutics) \u0026ndash; will provide effective and durable anticancer protection mediated by the immune system in dogs, locally and systemically. To our knowledge, this is the first report of a canine receiving intratumoral injections of a TLR4 agonist containing adjuvant EmT4\u0026trade; for a naturally occurring malignancy. Though EmT4\u0026trade; is an MPLA mimetic - as are some other adjuvants - it differs because it is a defined synthetic molecule and therefore should have fewer off-target effects than natural mixtures of agonists. GSK\u0026rsquo;s approved commercial product 3D-MPL is derived by hydrolyzing LPS from \u003cem\u003eSalmonella minnesota\u003c/em\u003e leading to the hydrolysis of any of the seven attached acyl chains and leaving a mixture of predominantly 4, 5, 6, and 7 acyl forms of the molecule. The one known to be active in humans is the hexaacyl form. The agonist in EmT4\u0026trade; is a synthetic form of this hexaacyl structure and has therefore effectively been in millions of people receiving vaccinations.\u003c/p\u003e"},{"header":"Case","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePatient\u003c/h2\u003e \u003cp\u003eAn 8-yr-old female spayed Boston terrier (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) belonging to one of the authors and who gave consent for treatment, was seen for a broad-based 3-cm soft tissue mass over the lateral side of the right elbow (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The lesion was non-painful and had been slowly enlarging for approximately 5 months. The mass was soft and fluctuant. During the observation period, it was aspirated for cytological evaluation on two separate occasions, yielding only adipose tissue each time. The mass was initially considered to be a lipoma.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDue to its large size and continued slow progression, the owner requested mass excision, electing to have the tumor injected prior to surgery with the adjuvant EmT4\u0026trade;, based on extensive personal scientific knowledge of TLR4 agonists and expertise and after conferring with other scientists and MDs. The findings by Baldrick \u003cem\u003eet al\u003c/em\u003e that showed a single high dose of MPLA in research dogs did not cause toxicity further underpinned this decision.\u003csup\u003e5\u003c/sup\u003e It was given IT two times separated by two weeks.\u003c/p\u003e \u003cp\u003eThe planned dose of EmT4\u0026trade; was based on data from related TLR4 agonists. EmT4\u0026trade; is similar to G100, which has been evaluated in murine models\u003csup\u003e16\u003c/sup\u003e and administered intratumorally (IT) repeatedly to human patients with follicular lymphoma with little to no toxicity.\u003csup\u003e12\u003c/sup\u003e Dogs receiving MPLA, a TLR4 agonist closely related to EmT4\u0026trade;, tolerated the injection well, \u003cem\u003ei.e.\u003c/em\u003e, without clinical adverse events and normal hematologic and biochemical screens, when given as a single-dose of 100 \u0026micro;g/kg, although repeated daily treatments caused signs of toxicity, further discussed in Materials and Methods (\u003cem\u003eEmT4\u0026trade; dose and injection schedule)\u003c/em\u003e.\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThe tumor grew after the first injection, from ~\u0026thinsp;3 cm to 5 cm diameter. The skin became taught over the tumor and was sensitive to touch and warm. Maximum growth occurred about 3\u0026ndash;6 hours after the injection. The dog experienced mild discomfort and lethargy, but was self-supporting, alert, responsive and ate and drank normally. These signs were compatible with a grade 1 Constitutional Signs adverse event according to the VCOG-CTCAE v.2.\u003csup\u003e17\u003c/sup\u003e We suspect that there was a spike in TNF-α within the tumor, and possibly systemically secondary to TLR4 activation. TNF-α is pyrogenic and could have induced fever although in this case body temperature wasn\u0026rsquo;t recorded at home. Fever would be consistent with the observation by Shi \u003cem\u003eet al\u003c/em\u003e that reported transient spikes of TNF-α in the blood correlated with the time of fever several hours after administration of the macrophage activator L-MTP-PE intravenously to dogs with osteosarcoma.\u003csup\u003e18\u003c/sup\u003e L-MTP-PE is also a TLR4 agonist.\u003csup\u003e19\u003c/sup\u003e Within 24 hours, the tumor decreased in size but remained enlarged although no longer painful. After the second injection two weeks later, the tumor did not enlarge appreciably, and the dog did not show signs of discomfort.\u003c/p\u003e \u003cp\u003eThe tumor underwent an excisional biopsy four weeks after the second IT injection, when size measurements were relatively unchanged. She recovered from surgery uneventfully. The tumor was fixed in formalin and embedded in paraffin wax for sectioning. The diagnosis was STS, grade 2. The margins showed tumor cells extending to the cut edge of the tumor indicating residual tumor remained at the site. The margins were later treated with two rounds of electrochemotherapy. There has not been recurrence to date, approximately 28 months following EmT4\u0026trade; treatment.\u003c/p\u003e \u003c/div\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eEmT4\u0026trade;\u003c/h2\u003e \u003cp\u003eEmT4\u0026trade; (\u003cem\u003eEmulsion with a Toll-Like Receptor 4 ligand\u003c/em\u003e) is an MPLA analog molecule in a squalene emulsion. EmT4\u0026trade; is produced by PAI Life Sciences, Inc (Seattle, WA) who provided it for this study. EmT4\u0026trade; was prepared by incorporating synthetic 3D (6-acyl)-PHAD into a squalene-based stable oil-in-water emulsion using established methods.\u003csup\u003e4,15\u003c/sup\u003e The emulsion contained squalene, glycerol, and polysorbate 80 as stabilizers, with a particle size around 100 +/- 40 nm as measured by dynamic light scattering. The final formulation was sterile filtered, aseptically filled into 2 mL vials, and stored at 2\u0026ndash;8\u0026deg;C until use. It was produced in an ISO7 GMP cleanroom following GLP protocols. It was tested post-production for sterility and particle size.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eEmT4™ dose and injection schedule\u003c/h3\u003e\n\u003cp\u003eTwo IT injections of EmT4\u0026trade; were given 2 weeks apart. The dose was deposited in several locations within the tumor by redirecting the needle without removing it. There were two separate IT injection sites for each treatment. The planned dose of EmT4\u0026trade; was based on data from related TLR4 agonists. EmT4\u0026trade; is similar to G100, which has been evaluated in murine models\u003csup\u003e16\u003c/sup\u003e and administered IT to human patients with follicular lymphoma at doses up to 40 \u0026micro;g weekly for eight weeks; adverse events were predominantly grade 1\u0026ndash;2.\u003csup\u003e12\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eAs mentioned above, MPLA - a compound closely related to EmT4\u0026trade; - was well tolerated in dogs at a single dose of 100 \u0026micro;g/kg indicating a wide safety margin for single-dose administration, although repeated daily dosing led to immune overstimulation and toxicity.\u003csup\u003e5\u003c/sup\u003e These data supported the conclusion that doses well below 100 \u0026micro;g per dog are likely to be safe. Following intratumoral injection, the amount of EmT4\u0026trade; retained within the tumor was estimated to be approximately 40 \u0026micro;g in a volume of ~\u0026thinsp;0.2 mL, well below doses associated with toxicity in dogs.\u003c/p\u003e\n\u003ch3\u003eHistopathology\u003c/h3\u003e\n\u003cp\u003eFormalin-fixed paraffin-embedded tumor tissue obtained via excisional biopsy was sectioned at 4 microns, stained with hematoxylin and eosin, and evaluated by two ACVP board certified pathologists independently at two different institutions, Louisiana State University and IDEXX Laboratories.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry\u003c/h2\u003e \u003cp\u003eImmunohistochemistry was performed for canine immune cell markers. The antibody clones used in this study have been previously utilized in published canine studies; specific references for each supports their use here. All IHC antibody markers have been tested in normal canine lymphoid tissue controls to confirm appropriate compartmental labeling.\u003c/p\u003e \u003cp\u003eThese include T cells (CD3, clone F7.2.38, Dako diluted 1:100)\u003csup\u003e20\u003c/sup\u003e, B cells (CD20, cat. #RB-9013-P, Thermo Scientific diluted 1:400)\u003csup\u003e21\u003c/sup\u003e, regulatory T cells (FoxP3, clone FJK-16S, Thermo Fisher diluted 1:100)\u003csup\u003e22\u003c/sup\u003e, macrophages, dendritic cells (Iba-1, FUJIFILM Wako Chemicals USA)\u003csup\u003e23\u003c/sup\u003e, and macrophages, histiocytes (CD204, clone SRA-E5, Abnova, diluted 1:200)\u003csup\u003e24\u003c/sup\u003e. Staining was done using the automated Leica BOND-MAX stainer according to published protocols.\u003csup\u003e25,26\u003c/sup\u003e\u003c/p\u003e \u003cp\u003ePrior to staining, heat-induced epitope retrieval was performed with a citrate-based pH 6.0 solution (Leica Biosystems) for 20 min at 100 C. Diaminobenzidine was used as the chromogen, positive stained cells appearing brown. Slides were scanned on the PhenoImager HT 2.0 (Akoya Biosciences) and QuPath software v0.6.0 was used for qualitative and quantitative analysis of positive staining cells.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eRNAscope™ In Situ Hybridization\u003c/h3\u003e\n\u003cp\u003eRNAscope\u0026trade; \u003cem\u003ein situ\u003c/em\u003e hybridization (ISH) was used to detect RNA transcripts for \u003cem\u003eCD4\u003c/em\u003e, \u003cem\u003eCD8A\u003c/em\u003e, \u003cem\u003eIFNG\u003c/em\u003e, \u003cem\u003eTNFA\u003c/em\u003e, and \u003cem\u003eIL-2\u003c/em\u003e within immune cells using FFPE tissue sections. The RNAscope\u0026trade; ISH assay (Advanced Cell Diagnostics-ACD, Newark, CA) was performed using the RNAscope\u0026trade; 2.5 LSx RED Reagent Kit on the automated BOND RXm platform (Leica Biosystems, Buffalo Grove, IL) as described previously.\u003csup\u003e26,27\u003c/sup\u003e Four-micron sections of FFPE tissues were mounted on positively charged Superfrost\u0026reg; Plus Slides (VWR, Radnor, PA, USA) and subjected to automated baking and deparaffinization, followed by heat-induced epitope retrieval using a ready-to-use EDTA-based solution (pH 9.0; Leica Biosystems) at 100\u0026deg; C for 15 min. Subsequently, tissue sections were treated with a ready-to-use protease (RNAscope\u0026trade; 2.5 LSx Protease, ACD) for 15 min at 40\u0026deg; C, followed by a ready-to-use hydrogen peroxide solution for 10 min at room temperature. Slides were then incubated with the ready-to-use probe mixture for 2 h at 40\u0026deg; C, and the signal was amplified using a specific set of amplifiers (AMP1 through AMP6 as recommended by the manufacturer). The signal was detected using a Fast Red solution for 10 min at room temperature. Finally, slides were counterstained with a ready-to-use hematoxylin stain for 5 min, followed by five washes with 1\u0026times; BOND Wash Solution (Leica Biosystems). Slides were rinsed in deionized water, dried in a 60\u0026deg; C oven for 30 min and mounted with Ecomount\u0026reg; (Biocare, Concord, CA, USA). Proprietary antisense probes were designed and provided by ACD using their algorithm for canine \u003cem\u003eCD4\u003c/em\u003e, \u003cem\u003eCD8A\u003c/em\u003e, \u003cem\u003eTNFA\u003c/em\u003e, \u003cem\u003eIFNG\u003c/em\u003e, and \u003cem\u003eIL-2\u003c/em\u003e (ACD catalog # 459558, 459548, 578948, 830438, and 1830858 for an IL-2 custom designed probe, respectively). Probe sequences from ACD are proprietary and not provided to users. Specific information on the probes including GenBank accession number, number of ZZ pairs designed, and target region of the mRNA are provided on the company\u0026rsquo;s website. On-target binding specificity of probes was verified using normal canine lymphoid tissue, \u003cem\u003ei.e.\u003c/em\u003e, lymph node, spleen, and tonsil as ISH controls. Slides were scanned on the PhenoImager HT 2.0 (Akoya Biosciences) and QuPath V0.6.0 was used for quantitative analysis of positive staining cells visualized as punctate red dots.\u003csup\u003e28\u003c/sup\u003e\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n\u003ch2\u003eHistopathology\u003c/h2\u003e\n\u003cp\u003eThe tumor was diagnosed as an STS, grade 2. Histopathologic examination revealed expansion of the subcutis by an unencapsulated, densely cellular and expansile neoplasm composed of neoplastic spindle cells arranged in streams and whorls and supported by a scant stroma (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC inset). Neoplastic cells exhibited moderate atypia and there were 14 atypical mitotic figures counted in 2.37mm\u003csup\u003e2\u003c/sup\u003e (equivalent to 10 high power fields). Intratumoral necrosis was not present in any of the examined sections. Together, the combined differentiation, mitotic, and tumor necrosis scores yielded a histologic total grade score of 4\u0026ndash;5, considered intermediate grade/grade 2.\u003csup\u003e29\u003c/sup\u003e Within multiple regions of the neoplasm, neoplastic cells were more widely separated by intratumoral interstitial edema, and within a given region, prominent lymphocytic cuffs delimit approximately 50% of blood vessels with sporadic infiltrating lymphocytes within other areas of the neoplasm (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC, \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e\n\u003cp\u003eThere was no pre-EmT4\u0026trade; injection sample available as multiple aspirates were non-diagnostic, and an interventional biopsy was not done given the slow growth of the tumor. Two archived FFPE tissue blocks at Louisiana Animal Disease Diagnostic Laboratory, Louisiana State University\u003c/p\u003e\n\u003cp\u003efrom two dogs with STS (right rear limb near the stifle and right forelimb lateral to the shoulder) of comparable histologies, grade, and cellular features are presented as examples of non-injected tumors in lieu of a pre-injected sample. However, these should not be construed as controls.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n\u003ch2\u003eImmunohistochemistry\u003c/h2\u003e\n\u003cp\u003eImmunohistochemistry for immune cell markers was performed on a selected FFPE tissue block representative of the treated STS, including CD3, CD20, FoxP3, CD204, and Iba-1. Four representative perivascular regions with large numbers of mononuclear cells were assessed and the proportion of cells immunolabeled by each specific IHC marker was determined. Within the cellular perivascular cuffs, CD20\u0026thinsp;+\u0026thinsp;B lymphocytes represented the most frequent cellular population, followed by CD3\u0026thinsp;+\u0026thinsp;T lymphocytes, Iba-1\u0026thinsp;+\u0026thinsp;macrophages/dendritic cells, CD204\u0026thinsp;+\u0026thinsp;macrophages/histiocytes, and FoxP3\u0026thinsp;+\u0026thinsp;regulatory T cells (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eLPS can activate B cells through paracrine signals as well as through B cell receptor binding of the associate carbohydrates \u0026ndash; thereby inducing cell proliferation and differentiation.\u003csup\u003e30\u003c/sup\u003e This may help to explain why there were large numbers of B cells in the clusters as EmT4\u0026trade; activates the same signaling cascade as LPS and should have similar effects on these cells. Abscopal effects are usually attributed to T cell-mediated immunity, but B cells also contribute through antibody production and cytokine signaling that modulate T cell activity. The presence of tertiary lymphoid structures rich in activated B cells correlates with better systemic and abscopal immune responses.\u003csup\u003e31\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eThe mean number of cells in four fields positive for T cells (CD3), regulatory T cells (FoxP3), B cells (CD20), macrophages/histiocytes (CD204), and macrophages/dendritic cells (Iba-1) are expressed as percentage of the total mononuclear cell population scanned (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e), as described.\u003csup\u003e25\u003c/sup\u003e Sporadic CD3\u0026thinsp;+\u0026thinsp;T lymphocytes and FoxP3\u0026thinsp;+\u0026thinsp;regulatory T lymphocytes were detected elsewhere within the neoplasm, while no CD20\u0026thinsp;+\u0026thinsp;B lymphocytes were identified beyond the cell clusters. Infiltration by CD204 positive histiocytes is widespread throughout the interstitium of the neoplasm, considered to be resident cells and not due to EmT4\u0026trade; treatment.\u003csup\u003e32\u003c/sup\u003e\u0026nbsp;\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003ePercent Positive in Immune Cell Clusters (mean)\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eBoston Terrier FS 8yr\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(patient)\u003c/strong\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eCD3\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eFoxP3\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eCD20\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eCD204\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eIba-1\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e24.6\u003c/p\u003e\n\u003cp\u003eSD 7.21\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e8\u003c/p\u003e\n\u003cp\u003eSD 1.48\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e57.2\u003c/p\u003e\n\u003cp\u003eSD 7.21\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e10.2\u003c/p\u003e\n\u003cp\u003eSD 4.55\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e36.2\u003c/p\u003e\n\u003cp\u003eSD 11.36\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eCatahoula Hound MC 8yr\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(archived tissue)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e18.53\u003c/p\u003e\n\u003cp\u003eSD 10.67\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e11.44\u003c/p\u003e\n\u003cp\u003eSD 3.93\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003eMixed Breed FS 11yr\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(archived tissue)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e10.32\u003c/p\u003e\n\u003cp\u003eSD 15.83\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9.46\u003c/p\u003e\n\u003cp\u003eSD 4.03\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eIHC slides were digitized and analyzed with digital pathology software (QuPath for positive cell detection and quantitative analysis). Four representative perivascular regions with large numbers of mononuclear cells were assessed for signal generated by each immune marker. The mean number of cells positive for T cells (CD3), regulatory T cells (FoxP3), B cells (CD20), and macrophages/dendritic/histiocytic cells (CD204, Iba-1) are expressed as percentage of the total mononuclear cell population scanned.\u003c/p\u003e\n\u003cp\u003eIn the two archived tumors that did not receive EmT4\u0026trade; injection, there were no cells positive for CD3, FoxP3, and CD20 lymphocytes (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). CD204\u0026thinsp;+\u0026thinsp;macrophages were similarly abundant and with a similar distribution in the injected and non-injected tumors. The marker Iba-1 that identifies macrophages and dendritic cells, differentiated between the injected tumor and the two non-injected tumors having approximately three times as many Iba-1 positive cells (36%) within perivascular regions compared to 9% and 11% for the non-injected tumors.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n\u003ch2\u003eRNAscope In Situ Hybridization (ISH)\u003c/h2\u003e\n\u003cp\u003eFour separate densely cellular, perivascular fields were examined for ISH signals and presented as the mean % positive cells (+/- SD) of the total cell count in the 4 fields analyzed (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab2\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003ePercent Cells Positive by ISH (mean)\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eCD4\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eCD8\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eTNF-\u0026alpha;\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eIFN-g\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eIL-2\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e29.00%\u003c/p\u003e\n\u003cp\u003eSD 6.18\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.20%\u003c/p\u003e\n\u003cp\u003eSD 1.09\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e23.55%\u003c/p\u003e\n\u003cp\u003eSD 6.43\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.31%\u003c/p\u003e\n\u003cp\u003eSD 0.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.12%\u003c/p\u003e\n\u003cp\u003eSD 0.084\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cem\u003eIn situ\u003c/em\u003e hybridization revealed that the highest proportion of positive transcripts were for \u003cem\u003eCD4\u003c/em\u003e and \u003cem\u003eTNFA\u003c/em\u003e at approximately 30% and 24%, respectively (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e). Although present, \u003cem\u003eCD8A\u003c/em\u003e-positive cells were appreciably less than \u003cem\u003eCD4\u003c/em\u003e, and \u003cem\u003eIFNG\u003c/em\u003e was detected at a low level. \u003cem\u003eIL2\u003c/em\u003e-positive cells were low in numbers.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e"},{"header":" Discussion and Conclusions","content":"\u003cp\u003eThis case study investigated tolerability and local immune responses following IT injection of a unique TLR4 agonist formulation in a canine STS. It is a case report that includes two archived non-injected canine tumors of comparable histology. Two independent attempts by the attending veterinarian to obtain a diagnostic cytologic sample by aspiration yielded only non-diagnostic adipose tissue - which is not uncommon for sarcomas that don\u0026rsquo;t always exfoliate when aspirated. For this reason, the mass was initially presumed to be a lipoma and surgical removal was considered an elective procedure at that time and not immediately done. The non-injected dog tumors with similar STS histologic features and grade as the treated tumor were utilized in lieu of the absence of a pre-injection sample. They served for comparison with post IT EmT4\u0026trade; but not as controls. Though not ideal, this was helpful to illustrate stark differences.\u003c/p\u003e\n\u003cp\u003eGenerally, canine STS do not exhibit pronounced immune responses due to the presumed low mutational burden resulting in fewer tumor antigens.\u003csup\u003e33\u003c/sup\u003e High grade, poorly differentiated canine STS on the other hand, likely have higher mutational burdens, and have been reported to be infiltrated with CD3, CD20, and FoxP3 lymphocytes to varying degrees. Myxosarcomas and perivascular wall tumors appear to be the most antigenic.\u003csup\u003e34\u003c/sup\u003e The STS from the dog of this report is considered intermediate grade based on moderate cell atypia and intermediate mitotic fraction, 14 atypical mitotic figures/2.37mm\u003csup\u003e2\u003c/sup\u003e (equivalent to 10 high power fields), without necrosis.\u003csup\u003e29\u003c/sup\u003e The archived STS from two dogs did not contain any lymphocytes within the tumor parenchyma. Avallone \u003cem\u003eet al\u003c/em\u003e reported that the canine fibrosarcomas examined in their study had no or low numbers of lymphocytes with counts statistically below those of myxosarcomas and perivascular wall tumors.\u003csup\u003e34\u003c/sup\u003e Without a pre-injection tissue sample to serve as a control, we cannot know what the cellular landscape was or even if there were already immune cell infiltrates present.\u003c/p\u003e\n\u003cp\u003eAfter the first EmT4\u0026trade; treatment but not the second, the dog was less active and preferred to lay down but alert and responsive the evening of the injection. After the first injection, but not the second, the tumor swelled rapidly and the overlying skin became taught, warm, and painful on touch, going from 3 to 5 cm. This is a classic feature of pseudoprogression, a hallmark of immune therapy, and was not considered tumor hyperprogression, since the tumor rapidly partially regressed in size a few days after swelling.\u003csup\u003e35\u003c/sup\u003e Activation of TLR4 receptors expressed by dendritic cells and macrophages within the tumor by EmT4\u0026trade; would result in release of proinflammatory cytokines like TNF-\u0026alpha; and chemokines. These would lead to the influx of immune cells and rapid swelling of the tumor due to edema and the volume of cells now occupying the sarcoma. The release of cytokines was confirmed by RNA hybridization with a TNF-\u0026alpha; probe showing transcripts in 23.6% of clustered immune cells.\u003c/p\u003e\n\u003cp\u003eTLR4 signaling is tightly regulated to prevent excessive inflammation and autoimmunity.\u003csup\u003e36\u003c/sup\u003e Consistent with clinical experience for many immunotherapies, adverse events are most prominent after initial dosing and are often milder or absent with subsequent administrations - including for TLR4 agonists due to autoregulation of strong initial innate responses.\u003csup\u003e36,37\u003c/sup\u003e In this study, the second dose of EmT4\u0026trade; did not elicit the adverse events observed after the first dose, yet remained immunostimulatory, as evidenced by TNF-\u0026alpha; transcripts detected in immune cells four weeks after the second injection. Given the short half-life of TNF-\u0026alpha; protein and transcripts in both humans and mice,\u003csup\u003e38\u003c/sup\u003e these findings indicate sustained immune activation up to the time of tumor excision. This pattern is consistent with a TLR4 regulatory model in which the first dose delivers a \u0026ldquo;danger signal and reset,\u0026rdquo; while the second dose provides immune instruction without triggering overt inflammation, potentially explaining the transient tumor enlargement observed after the initial treatment.\u003c/p\u003e\n\u003cp\u003eAlthough there was not a pre-injection sample for valid comparison, it is reasonable to conclude that these signs were associated with EmT4\u0026trade; due to the rapid response after injection of the formulation and compatible with acute inflammation that was not present pre-injection. Further, TNF-\u0026alpha; transcripts were found in the RNA hybridization assay implying its continual synthesis 4-weeks after the last EmT4\u0026trade; injection. The histologic features of this tumor \u0026mdash; absence of immune cells in the two non-injected, histologically comparable STS, presence of T cells, B cells, and macrophages within the injected tumor milieu forming dense perivascular cell aggregates \u0026mdash; taken together may suggest EmT4\u0026trade; IT induced a local inflammatory immune response. The infiltrates in the TME track closely with what has been described for murine and human tumors injected with related LPS mimetics or immunostimulatory compounds.\u003c/p\u003e\n\u003cp\u003ePerivascular cuffing by immune cells is a feature of some infectious diseases (\u003cem\u003ee.g.\u003c/em\u003e, canine distemper, rabies, etc). In cancer it is considered an immune response to the tumor.\u003csup\u003e39\u003c/sup\u003e Using murine models of human colorectal and pancreatic cancer, Stoltzfus \u003cem\u003eet al\u003c/em\u003e employed multimodal spatial analysis of immune cells clustered around intratumoral vasculature to investigate microanatomical organization of cellular infiltrates in unperturbed tumors and following immunotherapy. While perivascular immune cells were present in untreated tumors, following immunotherapy they were markedly increased.\u003csup\u003e39\u003c/sup\u003e The study also revealed that T cells co-localized with dendritic cells or activated macrophages in compact bundles. In our study, IHC revealed co-mingling of T and B cells with macrophages within the clusters (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). This may have been due to a direct effect of EmT4\u0026trade;; a pretreatment biopsy would have been conclusive that this was indeed the case. The finding of TNF-\u0026alpha; in immune cells using RNA hybridization supports the conclusion that the macrophages within clusters were activated.\u003c/p\u003e\n\u003cp\u003eStoltzfus \u003cem\u003eet al\u003c/em\u003e also reported that cell clusters were not found around all vessels, as was also the case in our observations where approximately 50% of tumor vasculature showed perivascular immune cell cuffs.\u003csup\u003e39\u003c/sup\u003e This raises interesting questions as to what explains the differences. Could there have been heterogeneity in the vasculature associated with cell clusters versus vessels not associated with cell clusters? Neovasculature within tumors can be disorganized and it is possible that there were differences in adhesion molecules expressed by endothelial cells that dictated which vessels would serve as entry points of immune cells into the tumor. This remains speculative and has yet to be determined.\u003c/p\u003e\n\u003cp\u003eGenerally, immune cells in the injected STS remained in thick perivascular clusters without dissemination into the deeper tumor. This may be due to where the EmT4\u0026trade; was deposited: Given the tumor\u0026rsquo;s large size and the small needle used for injection, EmT4\u0026trade; may not have been deposited deeply into the tumor parenchyma. In the two rodent models described by Stoltzfus \u003cem\u003eet al\u003c/em\u003e immune cells were not found in the deeper tumor tissue either.\u003csup\u003e39\u003c/sup\u003e Those tumors were not subjected to IT administration of the immunostimulants that the mice received. The investigators suggested immunosuppressive factors (\u003cem\u003ee,g.\u003c/em\u003e, hypoxia and others) may have prevented migration of immune cells from peripheral perivascular nests to the interior and is a possible explanation for what we saw. The persistence of dense layers of immune cells in perivascular spaces may be considered perivascular immune nests, immune-rich microenvironments capable of mediating immune effects \u003cem\u003ein situ\u003c/em\u003e.\u003csup\u003e39\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003ein situ\u003c/em\u003e RNA hybridization data provided insight into the function of the cell infiltrates. It suggests that EmT4\u0026trade; may not only have attracted lymphocytes to the TME but also activated immune function genes to produce transcripts for the proinflammatory cytokine TNF-\u0026alpha; in abundance. High levels of CD4 lymphocytes and TNF-\u0026alpha;, as described here, are hallmarks of a Th1 biased immune response. Seo \u003cem\u003eet al\u003c/em\u003e concluded that IT administration of the MPLA mimetic GLA-SE induced a Th1 response based on the predominance of CD4 lymphocyte infiltrates accompanied by relatively high concentrations of TNF-\u0026alpha; within human STS.\u003csup\u003e13\u003c/sup\u003e Compared to TNF-\u0026alpha;, IFN-g expression was low but nevertheless present in some cells in the post-EmT4\u0026trade; sample. The same can be said about CD8 positive cells with only\u0026thinsp;~\u0026thinsp;3% positivity. This may be due to the relatively short time interval (\u003cem\u003ei.e\u003c/em\u003e., 4 weeks) between the second EmT4\u0026trade; injection and tumor excision as immunotherapy often takes more than 8 weeks to induce pronounced cytotoxic effects.\u003csup\u003e40\u003c/sup\u003e It is therefore possible that had there been a longer observation period, there might have been a higher percentage of CD8 cells within the tumor. Notably, the human STS patients reported by Seo \u003cem\u003eet al\u003c/em\u003e received a total of 8 IT injections of GLA-SE \u0026ndash; one per week compared to 2 doses of EmT4\u0026trade; given to the dog of this report. There were also large numbers of B cells present in the lymphocyte clusters. The report by Venkataraman \u003cem\u003eet al\u003c/em\u003e confirmed LPS induces B cell proliferation and identified signaling pathways in B cells contributing to this response.\u003csup\u003e30\u003c/sup\u003e Expression of TLR4 in murine B cell lymphoma is important for the therapeutic activity of these agonists when given IT.\u003csup\u003e16\u003c/sup\u003e Additional studies are needed to confirm the direct effect EmT4\u0026trade; may have had in recruiting large numbers of B cells to the TME following IT administration to the dog\u0026rsquo;s STS. However, LPS is a potent stimulator of B cell proliferation and a role for EmT4\u0026trade; exerting a similar effect cannot be ruled out. The presence of B cells adds to the panoply of immune cells that appear to have trafficked into the TME in a coordinated response induced by the two IT EmT4\u0026trade; injections.\u003c/p\u003e\n\u003cp\u003eOnly low numbers of FoxP3 regulatory T lymphocytes were present supporting an active, not suppressive, inflammatory process. Myeloid-derived suppressor cells were not examined and merit further attention in the future. Interestingly, Richert \u003cem\u003eet al\u003c/em\u003e reported that a liposome formulation of a TLR4 agonist in their murine experimental osteosarcoma model could reverse the polarization of M2 macrophages shifting to an M1 phenotype, creating a proinflammatory TME.\u003csup\u003e19\u003c/sup\u003e Indeed, classically activated macrophages (M1-like) result from LPS or mimetic-triggered signaling through TLR4 and produce inflammatory cytokines that favor anti-tumor immune responses.\u003csup\u003e41\u003c/sup\u003e The ability of TLR4 agonists such as EmT4\u0026trade; to switch macrophage polarity from immunosuppressive to immunostimulatory phenotypes, alone or in combination therapy, is potentially an added benefit of IT administration of TLR4 agonists that prompts further investigation. Additional dogs with STS (or other histologies) are needed with biopsies pre-EmT4 IT injection, to conclude that EmT4 not only promotes tumor infiltration by immune cells, but it also activates them within the tumor parenchyma.\u003c/p\u003e\n\u003cp\u003eTumor antigens that are released by immunogenic cell death, are scavenged and processed by macrophages and dendritic cells that present tumor epitopes to T cells. The activated T cells migrate and multiply in draining lymph nodes expanding anti-tumor clonotypes that can circulate (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). These cells mediate MHC-restricted tumoricidal effects with specificity and can traffic back to the TME of the primary tumor as well as generating abscopal cytotoxicity at secondary tumor locations. Several publications have reported identical clones in the circulation and in the TME following IT administration of TLR4 agonists G100 and GLA-SE confirming this to be the case.\u003csup\u003e11,13\u003c/sup\u003e It will be interesting to determine if similar immunologic events occur following EmT4\u0026trade; IT treatment in canine cancer.\u003c/p\u003e\n\u003cp\u003eExpanded clinical studies with IT EmT4\u0026trade; are needed to pursue a long-term goal of combining it with other regimens in dogs with cancer. While it is unlikely that EmT4\u0026trade; would solely be used as a standalone therapy, it would be straightforward and immunologically sound to include it in combination with other established treatment strategies. As an example, low doses of radiation have an immunostimulatory effect, inducing immunogenic cell death releasing damaged-associated molecular patterns (DAMPs) that activate dendritic cells and macrophages as well as releasing tumor antigens and could be synergistic with the immune activation induced by EmT4\u0026trade;.\u003csup\u003e42\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eHuman patients in Merkel cell carcinoma and follicular lymphoma trials received low dose radiation treatment prior to IT TLR4 treatments.\u003csup\u003e11,12\u003c/sup\u003e Higher doses of radiation prior to TLR4 administration were used by Seo \u003cem\u003eet al\u003c/em\u003e treating high grade STS.\u003csup\u003e13\u003c/sup\u003e Treatment schedules often resemble those of palliative radiation, hypofractionation, which veterinary oncology has employed for many years and clients readily elect this form of treatment.\u003csup\u003e43,44\u003c/sup\u003e With further evidence, it would be convenient to add EmT4\u0026trade; to these treatments as part of clinical trials to further improve outcomes.\u003c/p\u003e\n\u003cp\u003eCombining TLR agonists with checkpoint inhibitors has proven to be an effective regimen in several human cancers.\u003csup\u003e45,46\u003c/sup\u003e With the advent of checkpoint inhibitors in veterinary oncology, IT EmT4\u0026trade; combined with an anti-PD1 antibody, anti-LAG3 antibody, or anti-CTLA-4 antibody could offer new opportunities to potentiate responses. Combination immunotherapy has been shown to be more effective than single agent.\u003csup\u003e47\u003c/sup\u003e Additionally, certain chemotherapeutic agents can induce immunogenic cell death including anthracyclines, certain platinum drugs, cyclophosphamide and others.\u003csup\u003e48\u003c/sup\u003e Cyclophosphamide given at low doses induces immunogenic cell death and can reduce the numbers of regulatory T cells.\u003csup\u003e49\u0026ndash;52\u003c/sup\u003e Combined with a TLR4 agonist such as IT EmT4\u0026trade;, the TME could be tipped in favor of Th1 biased anti-tumor immunity. Selective killing of regulatory T cells by low dose cyclophosphamide would enhance potent immune triggering by TLR4 activation.\u003csup\u003e50,52\u003c/sup\u003e Although in this study FoxP3\u0026thinsp;+\u0026thinsp;cells comprised only 8% of the intra-tumoral lymphocytes, the CD3/FoxP3 ratio is approximately 3. Compared with radiotherapy or monoclonal antibody treatments, the chemotherapy approach could be a novel, inexpensive, immunotherapeutic option when combined with IT TLR4 activation. It would likely be accepted by dog owners because of its simplicity and could be provided at a lower cost compared to surgery. Activating TLR4 with EmT4\u0026trade; could also potentially reduce the number of concurrent treatments with other costly immunotherapeutics by enhancing their efficacy when used in combinations.\u003c/p\u003e\n\u003cp\u003eKeeping in mind this is a case report of one dog, more experience with EmT4\u0026trade; is needed prospectively \u0026ndash; including pre-surgical biopsy \u0026ndash; to validate and extend the findings presented here. This single case study has provided valuable insight into a promising immune treatment that could potentially be scaled and find a place in the expanding veterinary immunotherapy quiver. Based on the dog in this report, EmT4\u0026trade; appears to be nontoxic and was simple and quick to administer, minimizing restraint and stress for the dog. For these reasons - as well as its powerful immune-stimulatory properties - acceptance by dog owners could become a reality, but only after future studies confirm and expand on the findings presented here.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTLR4\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etoll\u0026ndash;like receptor 4\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSTS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003esoft tissue sarcoma\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLPS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003elipopolysaccharide\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eEmT4\u0026trade;\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eEmulsion with a Toll\u0026ndash;Like Receptor ligand 4\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMPLA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emonophosphoryl lipid A\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIFN\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eg\u0026ndash;interferon gamma\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTNF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eα\u0026ndash;tumor necrosis factor alpha\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIL\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e2\u0026ndash;interleukin\u0026ndash;2\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTME\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etumor microenvironment\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e \u003cp\u003eThe dog owner provided consent and ethical considerations were discussed in detail by the dog owner and veterinarian, as well as with the other authors.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCompeting interests\u003c/strong\u003e \u003cp\u003eDC is the owner of PAI Life Sciences Inc. SG and JK are employees of PAI Life Sciences Inc., which holds intellectual property relevant to EmT4\u0026trade;, and produces EmT4\u0026trade;.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eCanine Cancer Alliance\u003c/p\u003e \u003cp\u003ePAI Life Sciences, Inc.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eSCH analyzed data and wrote the main manuscript with co-authors. SCH and MC prepared figures. MC performed the histologic, IHC, and ISH assays for the figures. JK helped with producing the tumor slices that were analyzed and assisted with developing the manuscript and interpreting the data. DRM provided veterinary care. SG and DC conceptualized the study. DC invented EmT4\u0026trade;, wrote the records used to produce the study article, and interpreted the data. All authors reviewed and discussed the data and edited the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eWe thank the Canine Cancer Alliance for its generous support of this study.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data generated or analyzed during this study are included in this published article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAznar MA, Tinari N, Rull\u0026aacute;n AJ, S\u0026aacute;nchez-Paulete AR, Rodriguez-Ruiz ME, Melero I. Intratumoral delivery of immunotherapy-Act locally, think globally. J Immunol. 2017;198(1):31\u0026thinsp;\u0026minus;\u0026thinsp;9. doi: 10.4049/jimmunol.1601145. PMID: 27994166.\u003c/li\u003e\n\u003cli\u003eTaleghani N, Bozorg A, Azimi A, Zamani H. Immunogenicity of HPV and HBV vaccines: adjuvanticity of synthetic analogs of monophosphoryl lipid A combined with aluminum hydroxide. 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PMID: 27031851; PMCID: PMC4984796.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"veterinary-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Veterinary Oncology](https://veterinaryoncology.biomedcentral.com/)","snPcode":"44356","submissionUrl":"https://submission.springernature.com/new-submission/44356/3","title":"Veterinary Oncology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"toll-like receptor 4, lymphocytes, monophosphoryl lipid A, lipopolysaccharide mimetic, immunogenic cell death, inflammatory cytokines, immunostimulatory","lastPublishedDoi":"10.21203/rs.3.rs-8982168/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8982168/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eImmunostimulatory adjuvants used in vaccines to protect against infectious disease have demonstrated efficacy in stimulating anti-cancer immunity. The most commercially advanced ones activate Toll-Like Receptor-4 (TLR4), a transmembrane signaling molecule expressed by macrophages and dendritic cells triggering innate immune responses. Lipopolysaccharide, the first identified TLR4 agonist, induces toxic, unregulated immune activation. Mimetics of monophosphoryl lipid A, the stimulatory component in lipopolysaccharide, reduced immunotoxicity while retaining immunostimulatory properties. Intratumoral injection of formulated TLR4 agonists can stimulate \u003cem\u003ein situ\u003c/em\u003e antitumor immune responses by recruitment of immune cells and production of inflammatory cytokines that exert antitumor effects via a variety of mechanisms \u0026ndash; including direct cancer cell death and recruitment of effector cells. Human clinical cancer trials have shown efficacy - both locally and through abscopal effects employing this approach.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eWe hypothesized that injection of a novel TLR4 agonist, EmT4\u0026trade;, into a canine soft tissue sarcoma (STS) could alter the tumor microenvironment by attracting and activating immune cells \u003cem\u003ein situ\u003c/em\u003e. With the dog owner\u0026rsquo;s interest and written consent, a 3-cm soft tissue mass on the right forelimb of an 8-year-old female spayed Boston terrier received two intratumoral injections of EmT4\u0026trade;, two weeks apart. There was transient lethargy on the day of the first injection that resolved within hours. The tumor was excised 4 weeks after the second injection. Histopathology, immunohistochemistry, and \u003cem\u003ein situ\u003c/em\u003e RNA hybridization were utilized to explore immune cell populations in the tumor microenvironment.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eHistopathology revealed grade 2 STS with large numbers of densely packed perivascular immune cells disseminated within the tumor. Immunohistochemistry for immune cell markers showed heterogeneous positive staining within cell clusters \u0026ndash; CD3 (25%), CD20 (57%), FOXP3 (8%), CD204 (5%), and Iba-1 (36%). \u003cem\u003eIn situ\u003c/em\u003e hybridization performed on serial STS sections with RNAscope\u0026trade; identified transcripts for CD4 (29%), TNF-α (24%), CD8 (3.2%), and interferon-g (1.3%) in lymphocyte clusters.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eEmT4\u0026trade; may have elicited an innate immune response that attracted and activated immune effector cells intratumorally. Clinical circumstances prevented acquisition of a pre-EmT4\u0026trade; biopsy hampering a definitive conclusion although cell infiltrates observed are unusual in canine STS. This case is foundational for continued EmT4\u0026trade; investigations for canine cancer immunotherapy.\u003c/p\u003e","manuscriptTitle":"Intratumoral injection of a novel TLR4 agonist elicits infiltration by immune effector cells in a canine soft tissue sarcoma: An Immunologic Case Study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-18 10:10:30","doi":"10.21203/rs.3.rs-8982168/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-24T15:41:30+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-11T01:31:10+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-06T07:14:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"125611061594009913966240077164948824309","date":"2026-03-19T16:11:56+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-18T19:01:03+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-18T06:58:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"269214411772509671823801721944709281426","date":"2026-03-18T03:13:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"256688084554393159339464552109706481633","date":"2026-03-17T23:43:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"37281611157629843686349801249130688043","date":"2026-03-17T14:25:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"201534173885761450644870976792323567126","date":"2026-03-16T16:35:48+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-16T14:16:52+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-11T04:49:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-11T04:48:49+00:00","index":"","fulltext":""},{"type":"submitted","content":"Veterinary Oncology","date":"2026-02-27T00:40:38+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"veterinary-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Veterinary Oncology](https://veterinaryoncology.biomedcentral.com/)","snPcode":"44356","submissionUrl":"https://submission.springernature.com/new-submission/44356/3","title":"Veterinary Oncology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4d5f7d4c-4894-496c-b92d-63042c4dfcda","owner":[],"postedDate":"March 18th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-04-24T15:55:27+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-18 10:10:30","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8982168","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8982168","identity":"rs-8982168","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}