Histology of companion dog tumors before and after IMAX treatment. A, Histology of dog RV soft tissue sarcoma. Left, perivascular lymphocytic infiltration (▲). Middle, lack of identifiable tumor cells and marked leukocytic (primarily neutrophils) infiltration with mitotically active giant cells (→). Right, perivascular lymphocytic infiltration (▲) and blue extracellular and intracellular granular materials (inset) located mainly at the tumor periphery. B, Histology of dog SP soft tissue sarcoma. Right, marked intratumoral lymphocytic infiltration (▲). C, Histology of dog CL mammary carcinomas. Ca, caudal mammary gland; Cr, cranial mammary gland. D, Histology of dog BM mammary carcinoma. Right, some areas of the tumor 3 weeks after the second treatment include both malignant epithelial (*) component and malignant mesenchymal component with osteoid (▲), characteristic of carcinosarcoma. Scale bars, 100 μm.

Immunohistochemistry (IHC) of RV soft tissue sarcoma 2 days post IMAX treatment

Antibodies used in IHC of canine tissues

Subject and tumor characteristics and IMAX treatments in companion dogs in the pilot study.

IMAX regimen for laboratory safety study in beagle dogs.

Naturally occurring canine invasive urothelial carcinoma (InvUC) often harbors a BRAFV595E mutation, analogous to human BRAFV600E found across multiple cancer types, and has been used to investigate the effects of BRAF-targeted therapy. To investigate proteomic and phosphoproteomic changes during therapy, we analyzed 42 samples from 14 dogs with BRAFV595E-positive InvUC treated with vemurafenib. Tumors were collected via cystoscopy before treatment (Pre-Vem), 1 month into treatment (Vem-1-month), and at the time of progressive disease (Vem-PD). We observed consistent early suppression of proteins and phosphosites in tumors after 1 month of vemurafenib treatment in dogs that responded to the treatment, followed by their rebound at the time of progressive disease, highlighting early tumor suppressive events that are later reversed as tumors adapt and develop resistance. Key pathways affected included "positive regulation of telomerase assembly and localization to Cajal body", "positive regulation of protein localization to telomere", and "positive regulation of protein localization to Cajal body". Additionally, consistent "Rho GTPase signaling" changes were observed across both proteomic and phosphoproteomic analyses, underscoring the functional reactivation of this pathway in resistant tumors. In summary, these findings reveal molecular signatures of early response and acquired resistance to vemurafenib and offer a valuable resource for future investigation of BRAF-targeted therapy.

Locally injected 2E'/PTX and IMAX show dose-dependent skin responses in mice

Objective:The goal was to determine the presence of a driver mutation in dogs with and without detectable cancer to set the stage for future cancer prevention research exploiting processes that inhibit cancer development in the presence of such mutations. The objective was to detect the v-raf murine sarcoma viral oncogene homolog B (BRAF) mutation BRAFV595E across the disease spectrum from non-neoplastic urothelium to invasive urothelial carcinoma (UC) in dogs. Methods:Scottish Terriers ≥ 6 years old with no outward evidence of urinary disease (n = 120) were screened for UC by urinalysis, urinary tract ultrasonography, follow-up cystoscopic biopsy, and necropsy when possible. Initial results were published in 2022. Extended follow-up is presented here. The BRAFV595E mutation was detected in urine sediment by droplet digital PCR and in bladder tissues by immunohistochemistry. Results:For 120 dogs screened, biopsy-confirmed UC was found in 41 dogs including 31 high-grade UC, 6 low-grade UC, 1 carcinoma in situ, and 3 dysplasias that progressed to high-grade UC. The BRAFV595E mutation was found in the urine of 23 of 26 dogs with high-grade UC, 4 of 5 dogs with low-grade UC, 2 of 3 dogs with dysplasia, and 12 of 66 dogs with no UC detected over a median of 14 months of follow-up (range, 4 to 71 months). The BRAFV595E mutation was detected in UC and non-neoplastic urothelium by immunohistochemistry. Conclusions:The BRAFV595E mutation is present in various stages of canine UC and non-neoplastic urothelium. This knowledge will contribute to future cancer prevention research. Clinical Relevance:Urothelial carcinoma screening should be implemented for Scottish Terriers and potentially dogs in other high-risk breeds. Biopsy and standardized imaging remain essential in UC management.

Locally injected IMAX does not alter systemic cytokine profiles reflecting minimal systemic absorption. Plasma levels of IFN-α, TNF-α, IL-1β, IL-10, CCL2, CCL5, CXCL1, and CXCL10 at 1 hour after subcutaneous or intratumoral injection of IMAX (containing 1 mg 2E′) in healthy and CT26 tumor-bearing BALB/c mice (7–8 weeks). n = 4 mice per group. Data are the mean ± SD. P value was calculated by Sidak multiple comparisons test, following two-way ANOVA.

Skin histology of healthy or tumor-bearing mice pre- and post-IMAX treatment (low mag)

Gram-stained section of blue granular material in the injection site (tumor)

Locally injected IMAX is retained at the injection site with minimal systemic absorption. PTX levels (μg/g) in skin (or tumor) and (ng/mL) in plasma at 6, 24, and 48 hours after subcutaneous or intratumoral injection of IMAX (containing 1 mg 2E′) in healthy and CT26 tumor-bearing BALB/c mice (7–8 weeks). n = 4 mice per time point. Data are the mean ± SD. P value was calculated by Sidak multiple comparisons test, following two-way ANOVA.

Locally injected IMAX shows dose-dependent skin response in mice. A, Scab formation at the injected site after dosing 0.2, 0.5, and 1 mg IMAX (2E′ equivalent) in healthy and CT26 tumor-bearing BALB/c mice (7–8 weeks). n = 3 (healthy mice) and 5 (tumor-bearing mice) per group. B, Average scab area over time. C, Area under the curves (AUC) of scab area vs. time plots in mice receiving different IMAX doses. P value was calculated by Sidak multiple comparisons test, following two-way ANOVA. Data are the mean ± SD.

Locally injected IMAX is retained at the injection site with minimal systemic absorption

Abstract Chemoimmunotherapy leverages the immune system to control tumor growth and develop durable antitumor immunity. With this goal, we previously developed a nanoparticulate immunoactive complex (IMAX), composed of polyethyleneimine–lithocholic acid conjugate, paclitaxel, and a cyclic dinucleotide, which showed robust antitumor activity in multiple mouse models following intratumoral administration. In this study, we evaluated the biodistribution of locally delivered IMAX and determined a safe and effective dose in mice to inform pilot safety and efficacy evaluations in dogs. In mice, IMAX remained localized at the injection site with minimal systemic exposure, induced rapid immune cell infiltration, evidenced by gross skin reactions and histologic changes, and achieved complete tumor regression even at one-fifth of the initial dose. Laboratory dogs tolerated IMAX well, with only transient, manageable adverse events. In a pilot study in companion dogs with naturally occurring cancer, IMAX administered at conservative doses induced histologic changes indicating immune cell infiltration and necrosis, accompanied by transcriptomic signatures of early innate immune pathway activation, resulting in measurable antitumor effects in the dogs with mammary carcinoma. These findings support the feasibility and safety of local IMAX therapy in tumor-bearing dogs and warrant further investigation with optimized dosing and combination strategies. Significance: Locally administered nanoparticulate IMAX induces complete tumor regression in mice and triggers rapid immune cell infiltration, while producing measurable antitumor responses in companion dogs with mammary carcinoma, supporting the translational potential.

Immune checkpoint blockade therapy targeting the PD-1/PD-L1 axis has shown remarkable clinical impact in multiple cancer types. However, despite its recent success, such impact has been shown to be limited to tumors encompassing specific tumor microenvironment characteristics. Furthermore, a significant proportion of initial responders eventually develop resistance. Combining PD-1/PD-L1 blockade with chemotherapy, radiotherapy, or targeted therapy have been suggested to overcome resistance, yet have been shown to be insufficient in fully accounting for resistance. Unlike normal, differentiated cells, most cancer cells produce large amounts of lactic acid. This metabolic property is often referred to as “aerobic glycolysis, ” a well-known metabolic reprogramming of cancer cells to sustain cell proliferation and a hallmark of cancer. Such property as well as others of the altered metabolism of cancer cells and its byproducts affect the anti-tumor immune response. Notably, glycolytic metabolites, such as lactate, regulate T cell proliferation and function. However, the mechanism behind how such metabolic alterations impact the cancer cells’ resistance to PD-1/PD-L1 blockade therapy remains unclear. Thus, we sought to decipher the role of tumor-cell derived lactic acid in PD-1/PD-L1 therapy resistance and propose new immunotherapeutic strategies to improve the efficacy of PD-1/PD-L1 blockade therapies. Here, we found that tumor cell-derived lactic acid renders the immunosuppressive tumor microenvironment in the PD-1/PD-L1 blockade-resistant tumors by inhibiting the interaction between the PD-L1 protein and anti-PD-L1 antibody. Furthermore, we showed that the combination therapy of targeting PD-L1 with our PD-L1 antibody-drug conjugate (PD-L1-ADC) and reducing lactic acid with the MCT-1 inhibitor, AZD3965, can effectively treat the PD-1/PD-L1 blockade resistant tumors. Altogether, the findings in this study uncover a new mechanism of how lactic acid induces an immunosuppressive tumor microenvironment and suggest a potential combination treatment strategy to overcome the tumor resistance to PD-1/PD-L1 blockade therapy and improve clinical outcomes. Alyssa Kim, Wonkyung Oh, Deepika Dhawan, Deborah W. Knapp, Seung-Oe Lim. The role of tumor microenvironment lactic acid in the cancer cell resistance to anti-PD-L1 and anti-PD-1 blockade therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4857.

Objective:To evaluate the RNA and protein expression of hypoxia-inducible factor (HIF-1α) in canine urothelial carcinoma (UC) compared to normal canine urinary bladder tissue. Methods:Dogs with normal urinary bladder tissue were enrolled at the time of euthanasia with the tissue obtained via necropsy within 1 hour after death. The high-grade UC tissue was collected via necropsy or cystoscopically utilizing a resectoscope. Dogs in the UC group were excluded if they were treated with chemotherapy or radiation therapy prior to tissue collection. Immunohistochemistry was performed on all tissues to evaluate intracytoplasmic and intranuclear immunoreactivity of HIF-1α using a semiquantitative immunoreactivity score (IRS). Ribonucleic acid sequencing was also performed to evaluate the expression of HIF-1α in normal urinary bladders and canine UC. Results:10 dogs with high-grade UC and 10 dogs with normal urinary bladder tissue were enrolled. The median intracytoplasmic HIF-1α in the UC group was mild in intensity with a low percentage of positive cells (median IRS, 1; range, 0 to 2). The control dogs had similar intracytoplasmic HIF-1α expression (median IRS, 1; range, 0 to 1). The difference in RNA expression of HIF-1α between groups was not significant (1.3-fold change). Conclusions:This study did not identify any differential RNA or protein expression of HIF-1α between normal urinary bladder tissue and UC in dogs. Clinical Relevance:HIF-1α is not differentially expressed in canine UC, but further exploration is necessary to evaluate if other proteins associated with hypoxia and angiogenesis could play a role in tumor growth and chemotherapy resistance in canine UC.

Naturally occurring canine invasive urinary carcinoma (iUC) closely resembles human muscle invasive bladder cancer in terms of histopathology, metastases, response to therapy, and low survival rate. The heterogeneous nature of the disease has led to the association of large numbers of risk loci in humans, however most are of small effect. There exists a need for new and accurate animal models of invasive bladder cancer. In dogs, distinct breeds show markedly different rates of iUC, thus presenting an opportunity to identify additional risk factors and overcome the locus heterogeneity encountered in human mapping studies. In the association study presented here, inclusive of 100 Shetland sheepdogs and 58 dogs of other breeds, we identify a homozygous protein altering point mutation within the NIPAL1 gene which increases risk by eight-fold (OR = 8.42, CI = 3.12–22.71), accounting for nearly 30% of iUC risk in the Shetland sheepdog. Inclusion of six additional loci accounts for most of the disease risk in the breed and explains nearly 75% of the phenotypes in this study. When combined with sequence data from tumors, we show that variation in the MAPK signaling pathway is an overarching cause of iUC susceptibility in dogs.

Correctly identifying perturbed biological pathways is a critical step in uncovering basic disease mechanisms and developing much-needed therapeutic strategies. However, whether current tools are optimal for unbiased discovery of relevant pathways remains unclear. Here, we create “Benchmark” to critically evaluate existing tools and find that most function sub-optimally. We thus develop the “Pathway Ensemble Tool” (PET), which outperforms existing methods. Deploying PET, we identify prognostic pathways across 12 cancer types. PET-identified prognostic pathways offer additional insights, with genes within these pathways serving as reliable biomarkers for clinical outcomes. Additionally, normalizing these pathways using drug repurposing strategies represents therapeutic opportunities. For example, the top predicted repurposed drug for bladder cancer, a CDK2/9 inhibitor, represses cell growth in vitro and in vivo. We anticipate that using Benchmark and PET for unbiased pathway discovery will offer additional insights into disease mechanisms across a spectrum of diseases, enabling biomarker discovery and therapeutic strategies. Multiple cellular pathways are altered in cancer and identifying them is relevant for prognosis and therapy. Here, the authors develop Benchmark and Pathway Ensemble Tool (PET), two computational approaches to optimise pathway discovery in cancer and predict related biomarkers and therapeutic avenues.