Tucatinib, a small molecule HER2 inhibitor, was approved in inoperable or metastatic HER2 + breast cancer. As many patients have tumors in challenging surgical locations, there is a need for imaging metrics to characterize tucatinib response and microenvironment impact. Molecular imaging can be used to quantify dynamic molecular changes that precede tumor size alterations and can target proliferation (fluorothymidine, [18F]-FLT), hypoxia (fluoromisonidazole, [18F]-FMISO) and HER2 expression ([89Zr]-Pertuzumab) with positron emission tomography (PET) imaging. The goal of this study is to non-invasively characterize tucatinib response in HER2 + breast cancer and quantify microenvironment modulation with advanced PET imaging. Mice with HER2 + human cell line (BT474) or patient derived xenograft (BCM 3472) tumors were treated with 50 mg/kg tucatinib and enrolled into imaging cohorts: imaged with [18F]-FLT-PET on days 0, 3 and 7, [18F]-FMISO-PET on days 0, 3 and 7, or [89Zr]Zr-Pertuzumab-PET on days 0 and 14. Proliferation, hypoxia and HER2 expression were quantified with standardized uptake value. A Mann–Whitney U Test assessed significance between groups. Tucatinib-treated human cell line and PDX tumors had significantly decreased hypoxia and proliferation relative to control tumors (p < 0.05). Tucatinib-treated BT474 tumors had significantly decreased HER2 expression (p < 0.05); however, no significant HER2 change was observed in BCM3472 tumors. Tucatinib significantly decreases intratumoral proliferation and hypoxia in both cell-line and patient-derived xenograft models of HER2 + breast cancer, which can be longitudinally quantified with PET imaging. Our data suggests molecular imaging may improve understanding and prediction of tucatinib response.

Rationale: Fibroblast activation protein (FAP) is a prominent transmembrane protein in the tumor microenvironment (TME) that promotes tumorigenesis. FAP has been a recent target for radionuclide-conjugated peptides for PET imaging and targeted radionuclide therapy (TRT). The objective of this study is to develop a theranostic approach using [ 64/67 Cu]-FAP2287 in combination with immunotherapy (IMT) to treat triple-negative breast cancer (TNBC). Methods 4T1 tumor-bearing BALB/c mice (N = 20) were injected and imaged with 100µCi [ 64 Cu]-FAP2287 PET at 1, 4, and 24h to quantify baseline FAP expression followed by autoradiography. 4T1-tumor bearing BALB/c mice (N = 42) were divided into Control, IMT, 800µCi [ 67 Cu]-FAP2287 or 4)[ 67 Cu]-FAP2287 + IMT and 1600µCi [ 67 Cu]-FAP2287, or [ 67 Cu]-FAP2287 + IMT (N = 5–10/group) and imaged with [ 64 Cu]-FAP2287 PET followed by treatment. All [ 67 Cu]-FAP2287 groups were imaged with SPECT at 4, 24, and 48h. Tumor growth and survival rates were monitored. Standard uptake values (SUV) quantified, statistical analysis and probability of survival were done with significance at p < 0.05. Results At baseline, there was a strong positive correlation between [ 64 Cu]-FAP2287 PET SUV Mean and autoradiography (R 2 = 0.7806,p < 0.001). No changes in [ 67 Cu]-FAP2287 retention over time was observed following the IMT. [ 64 Cu]-FAP2287 SUV Mean proved to be a reliable predictor of survival with 800µCi (R 2 = 0.84,p = 0.03) and 1600µCi (R 2 = 0.62,p = 0.01). FAP-TRT increased survival compared to controls and IMT. 1600µCi [ 67 Cu]-FAP2287 + IMT significantly decreased tumor volume (p = 0.0003) and increased survival (p = 0.0005) compared to [ 67 Cu]-FAP2287. Conclusion We can noninvasively target and predict response to FAP radiolabeled with elementally paired theranostic probe, [ 64/67 Cu]-FAP2287 in TNBC. IMT synergistically increased response to FAP-TRT and can guide personalized approaches to improve treatment efficacy.

The interest in 62Cu as a tracer for positron emission tomography (PET) has motivated several designs of a 62Zn/62Cu radionuclide generator, the most common of which is based on anion exchange resin and a mixed HCl/NaCl eluent. Since commercial 62Zn/62Cu generators are currently not available, the continued investigation of 62Cu-based radiopharmaceuticals relies on the implementation of in-house generator systems. This work presents a systematic evaluation of several key parameters relevant to anion exchange-based 62Zn/62Cu generators, including loading methodology, eluent composition, column dimensions, and 62Cu activity quantification. 62Zn and the long-lived tracer 65Zn were produced via proton irradiation of Cu targets and used to evaluate 62Zn/62Cu generator performance. A simplified target processing method was developed for generator manufacturing, representing a practical approach for handling irradiated targets given the 9.2 h half-life of 62Zn. Two generator loading approaches were compared, batch loading and fixed-bed loading. Fixed-bed loading yielded significantly lower zinc breakthrough than batch loading, with 1.1(2) × 10–3

Abstract Introduction: Due to lack of targeted therapies, triple-negative breast cancer (TNBC) accounts for 40% of breast cancer mortalities despite only comprising 10-15% of diagnoses. Combination of PD-1/PD-L1 inhibitors with radiation therapy have been shown to have synergistic effects in TNBC. Targeted radiotherapy (TRT), in which a targeting probe is attached to a cytotoxic radioisotope, has emerged as a promising technique to deliver a therapeutic radiation payload to cells expressing the protein of interest. This is advantageous when targeting proteins such as PD-L1, which is highly expressed in TNBC and strongly upregulated after radiation-induced cytotoxicity. The purpose of this study was to assess the efficacy and safety of a novel PD-L1 targeted radiotherapy to improve immunotherapy outcomes in TNBC. Methods: The PD-L1-targeted antibody atezolizumab was radiolabeled with lutetium-177, a cytotoxic radioisotope, to selectively deplete PD-L1+ cells. Athymic nude mice bearing high PD-L1-expressing MDA-MB-231 tumors were treated with saline (n=6), 250µCi (n=3) or 500µCi (n=5) of [177Lu]Lu-atezolizumab. Mice were imaged with Single Photon Emission Computed Tomography (SPECT) on days 1, 3, and 7 post-treatment, informing on distribution of the tracer in the tissue. Mice were monitored for 60 days for changes in tumor volume and body weight. Upon reaching endpoints, mice were euthanized and relevant organs (tumor, spleen, liver, kidney) were collected. Clearance organs were stained with hematoxylin and eosin to assess gross pathological changes. One- and two-way ANOVAs were utilized to assess differences between tumor volumes and SPECT imaging metrics across time, and log-rank tests were used to assess differences in survival between treatment groups. Results: Mice treated with 500µCi or 250µCi of [177Lu]Lu-atezolizumab demonstrated a significant reduction in tumor volume compared to control by day 20 (p < 0.05) or day 24 (p < 0.05) post-treatment, respectively. Increased median survival was observed in both treated groups (35 days in control versus 52 and 60 days in 250µCi and 500µCi treated mice, respectively, p < 0.01). No weight loss was observed in any treated groups, and histology revealed minimal splenic and renal toxicity, with mild to moderate hepatotoxicity consistent with reversible changes. SPECT imaging revealed high uptake of [177Lu]Lu-atezolizumab in the tumor by day 1 post treatment and high tumor to background ratios across all three imaging timepoints. Conclusions: Utilizing TRT to selectively deplete PD-L1+ cells resulted in decreased tumor volumes and increased survival in a mouse model of TNBC. Further, SPECT imaging validated high uptake of the PD-L1-targeted TRT within tumor tissue. These results validate [177Lu]Lu-atezolizumab as a novel PD-L1-targeted radiotherapy which has the potential to improve outcomes of immunotherapy in TNBC patients. Citation Format: Jonathan Moye, Hailey Houson, Sharmila Sridhar, Sherin James, Jason M. Warram, Suzanne E. Lapi, Anna G. Sorace, . PD-L1 targeted radiotherapy can reduce tumor burden and delay tumor growth in a mouse model of triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5819.

OBJECTIVE:This study aims to characterize and compare the saturation limits, spatial resolution, and image quality of various conventional and emerging positron-emitting radionuclides using a preclinical PET/CT scanner. By characterizing the performance of these radionuclides, the study sought to provide insights into their utility in high-resolution PET imaging. METHODS:Radionuclides (18F, 43Sc, 45Ti, 48V, 52Mn, 55Co, 64Cu, 68Ga, 89Zr) were evaluated on a GNEXT PET/CT scanner (Xodus Imaging, Torrance, CA) using saturation and Derenzo phantoms. Saturation was assessed by measuring the deviation between the actual and the region of interest (ROI) activity at varying concentrations of each radionuclide. Spatial resolution was quantified using full-width half maximum (FWHM) measurements from intensity profiles across six Derenzo phantom diameter sizes (1.2 mm-4.8 mm). Signal-to-noise ratios (SNRs) were calculated as a measure of image quality and Bland-Altman plots were used to assess the repeatability of resolution measurements. Statistical comparisons of test-retest were done to evaluate differences in accuracy and consistency across radionuclides. RESULTS:Saturation analysis revealed a broad range of limits across radionuclides, with 64Cu having the highest saturation threshold near 2 mCi (74 MBq), while 52Mn exhibited the lowest at approximately 250 μCi (9.25 MBq). Spatial resolution was inversely related to positron energy, with radionuclides like 18F and 64Cu producing clear images down to rod sizes of 1.6 mm compared to 68Ga and 55Co, which showed blurring at the same rod size. SNR analysis confirmed the superior image quality of lower-energy radionuclides, particularly for smaller structures, visually resolvable to 1.6 mm. Bland-Altman analysis showed that across the combination of rod sizes, 18F displayed improved repeatability in resolution measurements compared to 68Ga (standard errors of 0.03 and 0.15, respectively). CONCLUSION:This study demonstrates that the physical properties of radionuclides, particularly positron energy, significantly affected PET image quality, spatial resolution, and saturation thresholds. Lower-energy radionuclides like 18F and 52Mn are optimal for high-resolution applications, while higher energy radionuclides are better suited for high-activity imaging. These findings provide valuable guidance for optimizing radionuclide selection in preclinical and clinical PET imaging studies.

Two worlds exist for cupration of a ligand: the preparative lab where natural-abundance copper (in pure form) is typically used in excess versus the ligand, and the radiochemistry lab where the ligand is in great excess versus the copper radionuclide (unavoidably accompanied by contaminating metal ions). Here, seven synthetic free base water-soluble porphyrins have been examined to assess molecular designs suitable for targeted radiotheranostics. Studies were carried out with equimolar quantities of a porphyrin and natural abundance (nonradioactive) CuCl2 (10-4-10-7 M) and other metals in aqueous HEPES buffer (1 M) at room temperature (detection by absorption and fluorescence spectroscopy). Studies also were carried out with 64CuCl2 and excess porphyrin at 37 °C. Key results are as follows: (1) competition experiments with CoCl2, NiCl2, CuCl2, ZnCl2, FeCl2, and FeCl3 gave selective cupration. (2) The copper chelates of the porphyrins were stable in the aqueous solution for at least 24 h. (3) Copper porphyrins were obtained in apparent molar activities (AMAs) ∼6000 mCi/μmol (222 GBq/μmol) even in the presence of other metals. The results augur well for use of copper chelates of designer porphyrins in radiomedicine.

Rationale: Immune-targeted positron emission tomography (PET) allows for the non-invasive monitoring of immune populations with potential to provide early markers of response to novel immunotherapies in glioblastoma (GBM). Dynamic PET acquisitions can inform on tracer kinetics via dynamic modeling to enhance the GBM tumor microenvironment characterization, while its impact has yet to be understood. The objective of this study is to evaluate the quantitative description of dynamic granzyme B (GZP)-PET imaging and its ability to inform on combination immunotherapy response in orthotopic syngeneic GBM models. Methods: Orthotopic GBM murine models (GSC005-luc) were imaged with dynamic [64Cu]-NOTA-GZP PET (0-80 min) and T2 -weighted MRI one week post-treatment with saline or combination M002 virotherapy and anti-PD1 immunotherapy. One-, two-, and three-tissue compartment models were evaluated for suitability to tracer kinetics via Akaike information criterion. Biological validation consisted of ex vivo brain PET imaging, autoradiography, H&E and immunofluorescence for granzyme B. A subset of mice was longitudinally monitored via dynamic [64Cu]-NOTA-GZP PET at days 4 and 7. Changes in viable cell bioluminescence and radiological tumor volume (MRI) were used to determine response. Imaging-derived metrics including k1 , k2 , k3, k4 and SUV were evaluated across treatment and response groups via unpaired two-tailed T test. Results: Intratumoral [64Cu]-NOTA-GZP PET tracer kinetics showed improved fitting based on a two-tissue compartment model (p < 0.05). Intratumoral effector cell function (SUVmean TBR) and tracer binding rate (k3 ) were positively correlated with histological granzyme B density (p < 0.01). Increases in tumor tracer influx (k1 ) were observed in responders relative to non-responders (p < 0.01). Conclusions: Mathematical description of tracer kinetics via dynamic [64Cu]-NOTA-GZP PET offers complementary quantitative metrics for the characterization of immunotherapy response in GBM. Integration of dynamic protocols to immune-target PET approaches can provide clinically translatable metrics to differentiate immunotherapy-induced effects from tumor progression in GBM.

Positron-emitting cobalt-55 (t1/2 = 17.53 h, β+ = 77%, Eγ = 931.1 keV, Iγ = 75%) has the potential to be used for PET imaging and Auger electron-emitting cobalt-58m (t1/2 = 9.10 h, IC= 100%) has the potential to be used in targeted radionuclide therapy. Sarcophagines are a family of macrobicyclic cage amine ligands that form stable complexes with cobalt(III). This work investigates the potential of a derivative of sarcophagine with a pendent carboxylic acid functional group, 5-(8-methyl-3,6,10,13,16,19-hexaaza-bicyclo[6.6.6]icosan-1-ylamino)-5-oxopentanoic acid (known as MeCOSar), to serve as a ligand for cobalt-55. The cobalt(III) complex, [CoIII(MeCOSar)]3+, was synthesized and characterized as well as a complex where the MeCOSar ligand is conjugated to tumor-targeting peptide Tyr3-ocreotate, [CoIII(SarTATE)]3+. The MeCOSar and SarTATE ligands were radiolabeled with cobalt-55 to give [55Co][CoIII(MeCOSar)]n+ and [55Co][CoIII(SarTATE)] respectively and their biodistribution was evaluated in non tumor-bearing mice. The tumor uptake of [55Co][CoIII(SarTATE)] was then further evaluated in AR42J tumor-bearing mice and compared to the copper-64 complex, [64Cu][CuII(SarTATE)]. Radiolabeling SarTATE with cobalt-55 requires higher temperatures when compared to copper-64, and [55Co][CoIII(SarTATE)] has lower tumor uptake and higher liver uptake in tumor-bearing mice when compared to [64Cu][CuII(SarTATE)].

The human epidermal growth factor receptor 2 (HER2) is overexpressed in 15-30% of breast cancer cases, plays a significant role in pathogenesis, and has been used as a diagnostic indicator and therapeutic target. Herein, a bioconjugatable porphyrin chelator is described, bearing a water-solubilizing swallowtail motif that projects above and below the plane of the macrocycle. This porphyrin, in various quantities, was reacted in stochastic fashion with the lysine side chains of the anti-HER2 antibody trastuzumab to afford a series of porphyrin-trastuzumab (PorTraz) products with conjugation ratios of 2.3, 3.5, 4.1, and 5.2. Radiolabeling of the conjugates with 64Cu in HEPES buffer (pH 7.2) at 37 degrees C afforded a molar activity of up to 20 mu Ci/mu g (110 MBq/nmol). Binding was specific to the HER2 receptor, and uptake was observed for each conjugate. In mice bearing HER2+ BT-474 xenografts, PET imaging studies showed significantly higher tumor uptake, with a tumor standardized uptake value (SUV) of 0.95 +/- 0.08 versus 0.38 +/- 0.12 in the HER2- MDA-MB-468 xenograft control group at 24 h post-injection. PorTraz conjugates showed tumor SUV results of 1.25 +/- 0.31 for n = 2.3 porphyrin chelators per antibody and 0.59 +/- 0.20 for n = 5.2 porphyrin chelators per antibody. In summary, a series of porphyrin-trastuzumab conjugates were synthesized and labeled with 64Cu, displayed high binding to HER2+ cells, and illustrated uptake in mouse tumor xenografts observable by PET/CT imaging.

Theranostic strategies that utilize f-block therapeutic radionuclides, including 161Tb, 177Lu, 225Ac, and 227Th, suffer from a shortage of positron emission tomography (PET) imaging counterparts in the same chemical space and often rely on 68Ga as a surrogate. The 140Nd/140Pr in vivo PET generator, which belongs to the f-block, may address this issue and can be produced via the 141Pr-(p,2n)140Nd production route by using medium-energy cyclotrons. However, impurities in the target material, including stable Nd, and the inherent difficulty of adjacent lanthanide separations limit the achievable radionuclidic and chemical purity of 140Nd. In this work, we address these challenges through the purification and recycling of praseodymium target material and optimization of Nd/Pr separation. The resulting purified 140Nd was evaluated using DOTA and Macropa chelators via radiolabeling and in vitro stability studies. A target material purification and recycling method was developed for the monoisotopic 141Pr starting material to remove stable Nd impurities, yielding 90.3 ± 4.7% (n = 3) recovery. The purified 141Pr was isolated as Pr6O11 and irradiated with 24 MeV protons (20.07 MeV at the target surface) at 20 μA for 4 h, which produced 1417.0 ± 83.4 MBq (38.3 ± 2.2 mCi) of 140Nd at the end of bombardment (EOB). The produced 140Nd was purified through an optimized DGA normal method to recover 71.6 ± 6.3% pure 140Nd. The amount of stable Nd reduced progressively in each target purification cycle from >340 ppm without purification to <250 ppb after three cycles, while other measured metallic impurities were below 30 ppb. This improvement in target purity was reflected in the direct increase of apparent molar activity (AMA), when purified 140Nd was evaluated with DOTA and Macropa chelators. AMA of [140Nd]-Nd-DOTA and [140Nd]-Nd-Macropa increased from 70.3 MBq/μmol (1.9 mCi/μmol) and 74 MBq/μmol (2.0 mCi/μmol) to 8025.3 MBq/μmol (216.9 mCi/μmol) and 8473.0 MBq/μmol (229.0 mCi/μmol), respectively, after the third target purification cycle. Further evaluation of chelator-labeled 140Nd showed that [140Nd]-Nd-DOTA was stable in phosphate-buffered saline (PBS), saline, human serum, and mouse serum, whereas [140Nd]-Nd-Macropa was stable in all except human serum. This work established a practical methodological advance for the production of 140Nd/140Pr in vivo PET generators, combining optimized target recycling and radiochemical separation to enable scaled-up and high-molar activity 140Nd suitable for preclinical imaging. These advances support broader development of 140Nd/140Pr as a robust PET analogue, especially for f-block therapeutics.

Purpose This study investigates the potential of [47Sc]Sc-NOTA-trastuzumab as a novel targeted radionuclide agent in HER2 + breast cancer. Targeted radionuclide therapy offers a promising approach for enhanced delivery and internalization of radiation, minimizing off-target effects and treating advanced metastatic HER2 + breast cancers. By examining the synergistic relationship between [47Sc]Sc with trastuzumab, assessing [47Sc]Sc-NOTA-trastuzumab’s impact on immune function, and monitoring its passage of a compromised blood-brain barrier for targeting HER2 + breast-to-brain metastases, this study provides a path towards synergistic and precision radiotherapy in breast cancer metastasis. Experimental Design The stability, specificity and reactivity of [47Sc]Sc-NOTA-trastuzumab was characterized with high performance liquid chromatography, Lindmo, and cell binding assays. In vivo localization and therapeutic efficacy were assessed with SPECT imaging, biodistribution, and tumor monitoring in models of HER2 + breast cancer. Immunostaining against M1 macrophages was assessed to determine mechanisms of enhanced anti-tumoral immune activity. Results [47Sc]Sc-NOTA-trastuzumab demonstrates high specificity and serum stability in HER2 + cells. SPECT shows [47Sc]Sc-NOTA-trastuzumab has high specificity for HER2 + breast cancer cells in vivo in both the primary and metastatic setting. Longitudinally, tumors treated with [47Sc]Sc-NOTA-trastuzumab show therapeutic benefit and are comparable to those treated with 16x dose of trastuzumab alone, with no significant toxicity. Exploratory results show radiopharmaceutical uptake in a model of HER2 + brain metastasis. Increased M1-like macrophages were seen in HER2 + tumors relative to HER2- tumors following treatment with [47Sc]Sc-NOTA-trastuzumab. Conclusion [47Sc]Sc-NOTA-trastuzumab is serum stable and highly specific to HER2 + breast cancer. Developing and characterizing novel targeted radionuclide therapeutics has the potential to significantly improve radionuclide delivery in HER2 + metastatic disease.

The theranostic approach, which employs diagnostic radiopharmaceuticals to select patients who would benefit from targeted radiotherapy agents, has become an invaluable strategy for effective medical care. Scandium radionuclides offer the advantage of forming elementally matched and chemically identical diagnostic and therapeutic compounds, making them ideal candidates for this strategy. PSMA-617 is an established prostate-specific membrane antigen targeting agent and can be used as a proof of concept to investigate 43Sc, the diagnostic nuclide, and 47Sc, the therapeutic nuclide, as a theranostic pair. METHODS:Cellular uptake, competitive binding assays, and internalization studies were carried out using LNCaP or PC-3 cell lines. [43Sc]Sc-PSMA-617 was used in PET imaging studies in LNCaP or PC-3 tumor models, with time points ranging from 1-9 h. LNCaP tumor-bearing mice injected with [47Sc]Sc-PSMA-617 were imaged using SPECT up to 48 h. A longitudinal study was carried out using LNCaP tumor-bearing mice imaged with [43Sc]Sc-PSMA-617 prior to receiving a therapeutic dose of [47Sc]Sc-PSMA-617. RESULTS:43Sc and 47Sc were incorporated into PSMA-617 at radiochemical yields of >99%. Cellular uptake studies demonstrated high uptake and specificity to PSMA receptors for [47Sc]Sc-PSMA-617. In vivo PET studies showed specificity of [43Sc]Sc-PSMA-617 while SPECT studies demonstrated tumor retention of [47Sc]Sc-PSMA-617 up to 48 h. [47Sc]Sc-PSMA-617 demonstrated therapeutic efficacy by delaying tumor growth and increasing survival rates from a single administered dose in xenograft models. More importantly, the PET results from [43Sc]Sc-PSMA-617 PET were highly correlated with the therapeutic response from [47Sc]Sc-PSMA-617, showing that 43Sc PET data can predict therapeutic outcomes in individual animals from 47Sc agents, even in animals sharing a genetic background and implanted with tumors from the same cell line. CONCLUSIONS:Two chemically identical, PSMA-targeting radioscandium pharmaceuticals demonstrated in vivo stability, specificity and retention in PSMA+ tumor models. A theranostic study showed that a higher 43Sc PET SUVmean was strongly correlated to therapeutic response from the 47Sc agent, demonstrating that 43Sc and 47Sc can be used as an elementally matched theranostic pair.

Background:Mycoplasma genitalium (Mgen) is a sexually transmitted urogenital pathogen. Studies in sexual health clinic settings have demonstrated that Mgen infection, with similar prevalence to Chlamydia trachomatis infection, is becoming increasingly resistant to the limited classes of antibiotics with in vitro and clinical activity. Disulfiram (DS) is a Food and Drug Administration-approved drug for treatment of alcohol dependence that shows promise for repurposing as an antimicrobial agent. Methods:Minimum inhibitory concentration was performed via standard methods for human mycoplasmas and ureaplasmas as established by guideline M43-A of the Clinical and Laboratory Standards Institute. Mgen strain G37 was used as a part of a colorimetric checkboard assay to explore the mechanism of growth inhibition with DS, with emphasis on the role of copper on DS antimicrobial activity. Trace concentrations of radioactive 64Cu were used to understand Cu2+ trafficking and probe the antimicrobial mechanism of DS for strain G37 and 2 DS-resistant mutants (D3 and D4). Results:DS exhibits in vitro antimicrobial activity against Mgen isolates with low minimum inhibitory concentration values, regardless of susceptibility to other antibiotics. Growth inhibition studies with DS demonstrated that antimicrobial activity is copper dependent. Studies with 64Cu showed that DS greatly enhances copper uptake but that impaired DS-mediated copper uptake is not the mechanism for DS-resistance in Mgen mutants D3 and D4. Conclusions:DS exhibits potent copper-dependent antimicrobial activity against Mgen. DS or a similar compound could be developed as therapy for drug-resistant Mgen infections.

Abstract Breast cancer is known to have high levels of hypoxia, leading to poor prognosis and reduced treatment response. Sodium nitrite is an exogenous agent that has been shown to decrease hypoxia in ischemic limb injury and is being explored to improve hypoxia in the tumor microenvironment. The goal of this study is to use non-invasive imaging to characterize changes in perfusion, hypoxia, and drug delivery in vivo following exposure to sodium nitrite. Mouse models of breast cancer (BT474 human and 4T1 syngeneic cell lines) were treated with saline or sodium nitrite (165μg/kg IP) twice daily. Dynamic contrast-enhanced (DCE) MRI was used to monitor changes 20 minutes after the administration of a single dose of sodium nitrite. [18F]-fluoromisonidazole ([18F]FMISO) positron emission tomography (PET) enabled visualization and quantification of hypoxia within both models, with the BT474 model receiving one week of treatment and the 4T1 model receiving three doses of treatment before imaging. Sodium nitrite-induced changes to drug delivery were measured via dynamic [89Zr]Zr-atezolizumab PET in the 4T1 model, which informed on delivery alterations and the 24-hour retention of an antibody drug delivery. Mice were given sodium nitrite 24 hours, 12 hours, and 20 minutes prior to scan. Modeling of dynamic PET was performed with a two-tissue compartment model to extract biological metrics of perfusion (K1) and retention (k3). For all molecular studies, tracer uptake was quantified using the mean and distribution of the standardized uptake value (SUV) in the tumor. The Wilcoxon rank sum test was used to assess differences in Ktrans extracted from Kety-Tofts analysis of DCE-MRI data. One-way and two-way ANOVAs were used to determine the effects of treatment. Independent t-tests were used to examine differences between treatment groups. Mice treated with sodium nitrite showed increased perfusion (Ktrans from DCE-MRI) compared to those treated with saline (p=0.03). In both models, hypoxia was shown to be significantly reduced with the introduction of sodium nitrate compared to controls, with a decrease observed after 10 doses in BT-474 (p=0.01) and after two doses in 4T1 (p=0.02). Compartmental modeling showed that adding sodium nitrite increased the rate of delivery of the tracer (K1) compared to control (p=0.02) and improved retention (k3) at 24 hours (p=0.015). Non-invasive imaging revealed tumor microenvironment alterations in perfusion and oxygenation following sodium nitrite in two breast cancer models. Further, sodium nitrite was able to increase delivery of an antibody PET tracer, serving as a measure of antibody therapy delivery. As perfusion and hypoxia are key drivers in response to therapy, the addition of sodium nitrite can modulate hypoxia, perfusion, and drug delivery in solid tumors and could potentially offer new therapeutic insights for optimizing treatment in breast cancer. Citation Format: Kelsey M. O'Brien, Patrick N. Song, Katrina Ricart, Seth N. Lee, Chloe T. DeMellier, Zora Paschel, Urvi Rawal, Hailey Houson, Suzanne E. Lapi, Rakesh P. Patel, Anna G. Sorace. Sodium nitrite alters perfusion and hypoxia in preclinical models of breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2144.

Hypoxia in breast cancer leads to poorer prognosis and reduced treatment response. Sodium nitrite has demonstrated success as an endogenous and therapeutic mediator of nitric oxide (NO)-dependent signaling, especially in hypoxic environments. In this study, non-invasive imaging was used to test if nitrite therapy selectively increases perfusion, oxygenation, and drug delivery in mouse models of breast cancer (human BT474 or syngeneic 4T1 mouse models). Dynamic contrast-enhanced (DCE) MRI was used to monitor changes in perfusion, and [18F]-fluoromisonidazole ([18F]FMISO) positron emission tomography (PET) enabled quantification of changes in hypoxia after administration of nitrite. For all molecular studies, tracer uptake was quantified using the mean and distribution of the standardized uptake value (SUV) in the tumor. Sodium nitrite-dependent drug delivery to the tumor was measured via dynamic [89Zr]-atezolizumab PET. Modeling of dynamic PET was performed with a two-tissue compartment model to extract biological metrics of perfusion (K1) and retention (k3). Administration of a single i.p injection of nitrite (165μg/kg) significantly increased perfusion in 6/6 tumors by 91.5 ± 50.6% relative to saline. Moreover, hypoxia was significantly lower following nitrite treatment compared to saline controls. Compartmental modeling showed that adding nitrite increased the rate of delivery of [89Zr]-atezolizumab (K1) compared to control (p=0.02) and improved retention (k3) at 24 hours (p=0.015). As perfusion and hypoxia are key drivers in response to therapy, these results suggest sodium nitrite may be a viable candidate for modulating hypoxia, perfusion, and drug delivery in solid tumors for improving therapeutic outcomes.

Tumor-associated macrophages (TAMs) are a dominant immunosuppressive component of the tumor microenvironment (TME) in many solid cancers, including Lewis lung carcinoma (LLC). While immune checkpoint inhibitors (ICIs) primarily target adaptive immunity, the potential for small molecule checkpoint blockade to modulate the myeloid compartment remains poorly defined. Here, we evaluated the effects of a single-agent small molecule ICI on TAM phenotype and function in the LLC model, integrating molecular imaging with immune profiling. C57BL/6 mice bearing subcutaneous LLC tumors were treated with SRI-42475, a PD-1 targeting small molecule. To noninvasively monitor TAM polarization, longitudinal imaging was performed using a radiolabeled CD206-targeted probe before and after treatment. Imaging allowed for quantification of reduction of CD206 signal in treated tumors, consistent with a loss of M2-like macrophages. Flow cytometry at termination of the study provided an orthogonal assessment of therapy-induced shifts, including decreased expression of CD206 and increased expression of MHC-II and CD86, which indicates a reprogramming toward a pro-inflammatory phenotype. Macrophage remodeling was accompanied by enhanced infiltration and activation of CD8+ T cells within the TME. These findings demonstrate that small molecule checkpoint inhibition can reprogram the myeloid landscape in the absence of additional myeloid-targeting agents. Moreover, CD206-targeted imaging provides a valuable noninvasive biomarker of TAM dynamics and therapeutic response. Together, these results support further investigation of innate immune modulation by small molecule ICIs and highlight the translational utility of macrophage-directed imaging in immunotherapy. Rebecca J. Boohaker, Suzanne E. Lapi, Anna G. Sorace, Chloe Demellier, Volken Tekin, John Tillotson. Macrophage Reprogramming and Immune Checkpoint Blockade Efficacy in the Lewis Lung Carcinoma Model: Utility of longitudinal CD206-Targeted Molecular Imaging [abstract]. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr C048.