Comparison of postprocessing metrics in multimetabolic APT-weighted CEST and 2-deoxy-D-glucose-CEST-MRI for differentiating breast cancer subtypes in a murine model.

APT-weighted MRI Can Be an Early Marker for Demyelination.

Chemical exchange saturation transfer (CEST) imaging is a novel contrast mechanism, relying on the exchange between mobile protons in amide (-NH), amine (-NH2) and hydroxyl (-OH) groups and bulk water. Due to the targeted protons present in endogenous molecules or exogenous compounds applied externally, CEST imaging can respectively, generate endogenous or exogenous contrast. Nowadays, CEST imaging for endogenous contrast has been explored in pre-clinical and clinical studies. Amide CEST, also called amide proton transfer weighted (APT) imaging, generates CEST effect at 3.5 ppm away from the water signal and has been widely investigated. Given the sensitivity to amide proton concentration and pH level, APT imaging has shown robust performance in the assessment of ischemia, brain tumors, breast and prostate cancer as well as neurodegenerative diseases. With advanced methods proposed, pure APT and Nuclear Overhauser Effect (NOE) mediated CEST effects were separately fitted from original APT signal. Using both effects, early but promising results were obtained for glioma patients in the evaluation of tumor response to therapy and patient survival. Compared to amide CEST, amine CEST is also mobile proton concentration and pH dependent, but has a faster exchange rate between amine protons and water. The resultant CEST effect is usually introduced at 1.8-3 ppm. Glutamate and creatine, as two main metabolites with amine groups for CEST imaging, have been applied to quantitatively assess diseases in the central nervous system and muscle system, respectively. Glycosaminoglycan (Gag) as a representative metabolite with hydroxyl groups has also been measured to evaluate the cartilage of knee or intervertebral discs in CEST MRI. Due to limited frequency difference between hydroxyl protons and water, 7T for better spectral separation is preferred over 3T for GagCEST measurement. The applications of CEST MRI with exogenous contrast agents are still quite limited in clinic. While certain diamagnetic CEST agents, such as dynamic-glucose, have been tried in human for brain tumor or neck cancer assessment, most exogenous agents, i.e., paramagnetic CEST agents, are still tested in the pre-clinical stage, mainly due to potential toxicity. Engineered tissues for tissue regeneration and drug delivery have also shown a great potential in CEST imaging, as many of them, such as hydrogel and polyamide materials, contain mobile protons or can be incorporated with CEST specific chemical compounds. These engineered tissues can thus generate CEST effect in vivo, allowing a possibility to understand the fate of them in vivo longitudinally. Although the CEST MRI with engineered tissues has only been established in early stage, the obtained first evidence is crucial for further optimizing these biomaterials and finally accomplishing the translation into clinical use.

PS3-08: Assessment of early response to neoadjuvant systemic therapy (NAST) of triple-negative breast cancer (TNBC) using chemical exchange saturation transfer (CEST) MRI: A pilot study

Chemical exchange saturation transfer (CEST) magnetic resonance imaging (MRI) can probe tissue biochemistry in vivo with high resolution and sensitivity without requiring exogenous contrast agents. Applying CEST MRI at ultrahigh field provides advantages of increasing spectral resolution and improving sensitivity to metabolites with faster proton exchange rates such as glutamate, a critical neurotransmitter in the brain. Prior magnetic resonance spectroscopy and CEST MRI studies have revealed altered regulation of glutamate in patients with multiple sclerosis (MS). While CEST imaging facilitates new strategies for investigating the pathology underlying this complex and heterogeneous neurological disease, CEST signals are contaminated or diluted by concurrent effects (e.g., semi-solid magnetization transfer (MT) and direct water saturation) and are scaled by the T1 relaxation time of the free water pool which may also be altered in the context of disease. In this study of 20 relapsing-remitting MS patients and age- and sex-matched healthy volunteers, glutamate-weighted CEST data were acquired at 7.0 T. A Lorentzian fitting procedure was used to remove the asymmetric MT contribution from CEST z-spectra, and the apparent exchange-dependent relaxation (AREX) correction was applied using an R1 map derived from an inversion recovery sequence to further isolate glutamate-weighted CEST signals from concurrent effects. Associations between AREX and cognitive function were examined using the Minimal Assessment of Cognitive Function in MS battery. After isolating CEST effects from MT, direct water saturation, and T1 effects, glutamate-weighted AREX contrast remained higher in gray matter than in white matter, though the difference between these tissues decreased. Glutamate-weighted AREX in normal-appearing gray and white matter in MS patients did not differ from healthy gray and white matter but was significantly elevated in white matter lesions. AREX in some cortical regions and in white matter lesions correlated with disability and measures of cognitive function in MS patients. However, further studies with larger sample sizes are needed to confirm these relationships due to potential confounding effects. The application of MT and AREX corrections in this study demonstrates the importance of isolating CEST signals for more specific characterization of the contribution of metabolic changes to tissue pathology and symptoms in MS.

To investigate the effects of frequency drift on chemical exchange saturation transfer (CEST) imaging at 3T, and to propose a new sequence for correcting artifacts attributed to B0 drift in real time. A frequency-stabilized CEST (FS-CEST) imaging sequence was proposed by adding a frequency stabilization module to the conventional non-frequency-stabilized CEST (NFS-CEST) sequence, which consisted of a small tip angle radiofrequency excitation pulse and readout of three non-phase-encoded k-space lines. Experiments were performed on an egg white phantom and 26 human subjects on a heavy-duty clinical scanner, in order to compare the difference of FS-CEST and NFS-CEST sequences for generating the z-spectrum, magnetization transfer ratio asymmetry (MTRasym ) spectrum, and amide proton transfer weighted (APTw) image. The B0 drift in CEST imaging, if not corrected, would cause APTw images and MTRasym spectra from both the phantom and volunteers to be either significantly higher or lower than the true values, depending on the status of the scanner. The FS-CEST sequence generated substantially more stable MTRasym spectra and APTw images than the conventional NFS-CEST sequence. Quantitatively, the compartmental-average APTw signals (mean ± standard deviation) from frontal white matter regions of all 26 human subjects were -0.32% ± 2.32% for the NFS-CEST sequence and -0.14% ± 0.37% for the FS-CEST sequence. The proposed FS-CEST sequence provides an effective approach for B0 drift correction without additional scan time and should be adopted on heavy-duty MRI scanners.

BackgroundAmide proton transfer (APT), a specific type of chemical exchange saturation transfer (CEST) MRI, has proved valuable in tumor diagnosis and characterization by detecting mobile protein/peptides in cancerous tissues. However, T1 confounds CEST measurements, leading to reduced specificity to amides and potential misinterpretation of APT imaging.PurposeThe study aimed to investigate the feasibility of the quasi-steady-state (QUASS)-based apparent exchange-dependent relaxation (AREX) analysis in correcting T1 for unbiased tumor APT MRI at 3T.Materials and MethodsCEST MRI experiments were conducted on an egg white phantom and on prospectively enrolled brain tumor patients with T1 values modulated by gadolinium (Gd). QUASS algorithm was employed to reconstruct steady-state Z spectra. Conventional T1-uncorrected CEST effect was quantified with a multipool Lorentzian function from QUASS Z spectra. The non-QUASS AREX and QUASS-based AREX with T1 correction were calculated from the inverse of non-QUASS and QUASS Z spectra, respectively. The student’s t-test and Bland-Altman plots were performed to assess the statistical difference and consistency between pre- and post-Gd measurements.ResultsIn the phantom study, vials with different T1 values showed conspicuous discrepancy on the conventional uncorrected APT and non-QUASS AREX maps, but comparable contrast on the QUASS-based AREX map. In the human study, 13 patients were enrolled. The contralateral normal-appearing white matter exhibited no substantial change in T1 and similar CEST effect between uncorrected APT, non-QUASS AREX, and QUASS-based AREX pre- and post-Gd (all P > .05). However, the tumor regions showed significantly reduced T1 post-Gd that altered the CEST measurements obtained from uncorrected APT and non-QUASS AREX (both P < .001). In comparison, QUASS-based AREX measurements were in excellent agreement between pre- and post-Gd (P = .19).ConclusionQUASS-based AREX analysis can effectively correct T1 contamination in CEST measurements, facilitating unbiased tumor APT MRI at 3T.

To quantify chemical exchange saturation transfer contrast in upper extremities of participants with lymphedema before and after standardized lymphatic mobilization therapy using correction procedures for B0 and B1 heterogeneity, and T1 relaxation. Females with (n = 12) and without (n = 17) breast cancer treatment-related lymphedema (BCRL) matched for age and body mass index were scanned at 3.0T MRI. B1 efficiency and T1 were calculated in series with chemical exchange saturation transfer in bilateral axilla (B1 amplitude = 2µT, Δω = ±5.5 ppm, slices = 9, spatial resolution = 1.8 × 1.47 × 5.5 mm3 ). B1 dispersion measurements (B1 = 1-3 µT; increment = 0.5 µT) were performed in controls (n = 6 arms in 3 subjects). BCRL participants were scanned pre- and post-manual lymphatic drainage(MLD) therapy. Chemical exchange saturation transfer amide proton transfer(APT) and nuclear Overhauser effect (NOE) metrics corrected for B1 efficiency were calculated, including proton transfer ratio(PTR'), magnetization transfer ratio asymmetry , and apparent exchange-dependent relaxation (AREX'). Nonparametric tests were used to evaluate relationships between metrics in BCRL participants pre- versus post-MLD (two-sided P < 0.05 required for significance). B1 dispersion experiments showed nonlinear dependence of Z-values on B1 efficiency in the upper extremities; PTR' showed < 1% mean fractional difference between subject-specific and group-level correction procedures. PTR'APT significantly correlated with T1 (Spearman's rho = 0.57, P < 0.001) and body mass index (Spearman's rho = -0.37, P = 0.029) in controls and with lymphedema stage (Spearman's rho = 0.48, P = 0.017) in BCRL participants. Following MLD therapy, PTR'APT significantly increased in the affected arm of BCRL participants (pre- vs. post-MLD: 0.41 ± 0.05 vs. 0.43 ± 0.03, P = 0.02), consistent with treatment effects from mobilized lymphatic fluid. Chemical exchange saturation transfer metrics, following appropriate correction procedures, respond to lymphatic mobilization therapies and may have potential for evaluating treatments in participants with secondary lymphedema.

Chemical exchange saturation transfer (CEST) MRI is a non-invasive molecular imaging technique with potential applications in pre-clinical and clinical studies. Applications of amide proton transfer-weighted (APT-w), glutamate-weighted (Glu-w) and creatine-weighted (Cr-w) CEST, among others, have been reported. In general, CEST data are acquired at multiple offset-frequencies. In reported studies, different offset-frequency step sizes and interpolation methods have been used during B0 inhomogeneity correction of data. The objective of the current study was to evaluate the effects of different step sizes and interpolation methods on CEST value computation. In the current study, simulation (Glu-w, Cr-w and APT-w) and experimental data from the brain were used. Experimental CEST data (Glu-w) were acquired from human volunteers at 7 T and brain tumor patients (APT-w) at 3 T. During B0 inhomogeneity correction, different interpolation methods (polynomial [degree-1, 2 and 3], cubic-Hermite, cubic-spline and smoothing-spline) were compared. CEST values were computed using asymmetry analysis. The effects of different step sizes and interpolation methods were evaluated using coefficient of variation (CV), normalized mean square error (nMSE) and coefficient of correlation parameters. Additionally, an optimum interpolation method for APT-w values was selected based upon fitting accuracy, T-test, receiver operating characteristic analysis, and its diagnostic performance in differentiating low-grade and high-grade tumors. CV and nMSE increase with an increase in step size irrespective of the interpolation method (except for cubic-Hermite and cubic-spline). The nMSE of Cr-w and Glu-w CEST values were least for polynomial (degree-2 and 3). The quality of Glu-w CEST maps became coarse with the increase in step size. There was a significant difference (P < .05) between low-grade and high-grade tumors using polynomial interpolation (degree-1, 2 and 3); however, linear interpolation outperforms other methods for APT-w data, providing the highest sensitivity and specificity. In conclusion, depending upon the saturation parameters and field strength, optimization of step size and interpolation should be carried out for different CEST metabolites/molecules. Glu-w, Cr-w and APT-w CEST data should be acquired with a step size of between 0.2 and 0.3 ppm. For B0 inhomogeneity correction, polynomial (degree-2) should be used for Glu-w and Cr-w CEST data at 7 T and linear interpolation should be used for APT-w data at 3 T for a limited frequency range.

To determine the optimal saturation power for chemical exchange saturation transfer (CEST) imaging and evaluate the prognostic value of CEST parameters at different saturation powers in patients with acute ischemic stroke (AIS). Seventy-nine AIS patients underwent CEST imaging at saturation powers of 1, 1.5, and 2 μT. Amide proton transfer (APT#) and nuclear overhauser enhancement (NOE#) signals were quantified using the numerical fitting of the extrapolated semi-solid magnetization transfer reference (NEMR) method and compared to conventional amide proton transfer-weighted (APTw) signals based on the magnetization transfer ratio asymmetry at 3.5 ppm. Infarction visibility and hypointense artifacts on APT#, NOE#, and APTw images were graded on a three-point scale and compared using the Friedman test. Independent t-tests were used to compare CEST parameters between patients with favorable and unfavorable outcomes at 90 days. Hypointense artifacts were most pronounced at 2 μT and minimized at 1 μT on APT# and NOE# images (p < 0.001). Infarctions were best visualized at 1 μT in 78.6% and 64.3% of patients on APT# and NOE# images, respectively, whereas APTw images provided poor lesion visibility at all saturation powers. Ischemic lesions showed decreasing APT# and NOE# values with increasing saturation power. Patients with favorable outcomes had significantly higher APT# values and smaller percent change of APT# at 1 μT compared to those with unfavorable outcomes (p < 0.05). APT# images at 1 μT provided superior image quality and demonstrated a significant correlation with 90-day neurological outcomes in AIS patients. Question Standardized scanning parameters for chemical exchange saturation transfer (CEST) imaging in the evaluation of acute ischemic stroke (AIS) have not yet been established. Findings Amide proton transfer (APT#) images acquired with 1-μT saturation power provided optimal image quality and were significantly associated with 90-day neurological outcomes in AIS patients. Clinical relevance Scanning parameters affect image contrast and the CEST effect. Our study supports the use of 1-μT saturation power for improved diagnostic quality and prognostic evaluation, contributing to the standardization of CEST imaging in clinical stroke assessment and future research.

Stereotactic radiosurgery for the treatment of brain metastases delivers a high dose of radiation with excellent local control, but increases the likelihood of radiation necrosis. As shown in our previous work, saturation transfer MRI, consisting of quantitative magnetization transfer (qMT) and chemical exchange saturation transfer (CEST), is a promising technique for distinguishing radiation necrosis (RN) from tumour progression (TP) in brain metastases. A 3D qMT/CEST acquisition was recently implemented and over 100 patients have been scanned to date. The purpose of this work is to assess the ability of advanced MRI parameters, including qMT and CEST metrics, which are sensitive to macromolecules and metabolism. The specific metrics that were explored included the amide and NOE contributions of the magnetization transfer ratio (MTR), the MTR asymmetry, the apparent exchange-dependent relaxation (AREX), the qMT semi-solid pool fraction and the T1 and T2 relaxation times. For a subset of the patients, dynamic susceptibility contrast (DSC) perfusion images were acquired. Examples of confirmed tumour progression and radiation necrosis cases will be presented, comparing the structural images (pre- and post-contrast T1-weighted and FLAIR images) with parameter maps from qMT and CEST and also the relative cerebral blood flow (rCBF) from DSC perfusion imaging. Interim cohort results will be presented. Approaches for standardizing the parameters across multiple MRI vendors are also explored.

Relaxation-compensated chemical exchange saturation transfer MRI in the cervical spinal cord at 3T: An application in multiple sclerosis

Background: Immune checkpoint blockades (ICB) improve outcomes of patients with some solid tumors such has melanoma and lung cancer, however the efficacy of ICB alone showed limited efficacy in many other tumors such as breast cancer. Radiation therapy is a promising combination partner of ICB as a strong immune stimulator so called in situ tumor vaccine, however it also can increase immune suppressive repertoires. TIGIT (T cell Immunoglobulin and ITIM domain) is an inhibitory receptor expressed on activated T cells and NK cells. We evaluated the effects of TIGIT blockade in addition to local RT and PD-1 blockade as a strategy to overcome therapeutic resistance of ICI in a murine breast cancer model. Materials and Methods: 4T1-Luc tumors were treated with various strategies including control, RT, anti-PD-1, anti-TIGIT, RT+anti-PD-1, RT+anti-TIGIT, anti-PD-1+anti-TIGIT, and RT+anti-PD-1+anti-TIGIT. A total of 24 Gy in 3 fractions at 1 week was delivered to the tumor at hindlimb (primary tumor) while the tumor at flank (secondary tumor) was left unirradiated. 10 mg/kg of anti-PD-1 blocking antibodies and 10 mg/kg of anti-TIGIT blocking antibodies were intraperitoneally injected for 6 times for 2 weeks. Flow cytometry, IHC staining, and ELISA were performed to assess the immunologic status after treatments. Results: The triple combination therapy (TCT) group showed the most superior growth delay of primary and secondary tumor growth among treatments. The number of metastatic lung nodule was significantly reduced by TCT as well. Plasma levels of interferon-β and interferon-γ were the highest after TCT. Moreover, TCT significantly increased the proportion of splenic CD8+ dendritic cells among myeloid cells, and effector-memory (CD44+CD62L−) cells among splenic Foxp3− non-regulatory CD4+ and CD8+ T cells. Furthermore, expression of CD226, which is an activating counter-receptor of TIGIT, on splenic CD8+ T cells was elevated by TIGIT blockade, possibly suggesting the activation of systemic immune response by addition of TIGIT blockade to local RT and PD-1 blockade. Meanwhile, TCT decreased splenic Foxp3+ regulatory T cells, as well as the expression of CD39 on splenic Foxp3+ regulatory T cells. The increase of CD8+ T cell infiltration in primary and secondary tumors was most prominent in TCT group. Conclusion: TIGIT blockade elicits systemic immune responses in a murine triple-negative breast cancer model. when combined with local RT and PD-1 blockade, which were correlated with significant delay in the growth of both irradiated and unirradiated tumors. These results suggest that TIGIT blockade could be a viable approach to increase the efficacy of RT and immune checkpoint inhibitors in breast cancer which is a relatively immunologically cold tumor. (Work supported by a grant from National Research Foundation of Korea #2023R1A2C3003782 to In Ah Kim). Citation Format: Seongmin Kim, Seung Hyuck Jeon, In Ah Kim. TIGIT blockade combined with local radiotherapy and PD-1 blockade in a murine triple-negative breast cancer model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1360.

Chemical exchange saturation transfer (CEST) imaging of fast exchanging amine protons at 3ppm offset from the water resonant frequency is of practical interest, but quantification of fast exchanging pools by CEST is challenging. To effectively saturate fast exchanging protons, high irradiation powers need to be applied, but these may cause significant direct water saturation as well as non-specific semi-solid magnetization transfer (MT) effects, and thus decrease the specificity of the measured signal. In addition, the CEST signal may depend on the water longitudinal relaxation time (T1w ), which likely varies between tissues and with pathology, further reducing specificity. Previously, an analysis of the asymmetry of saturation effects (MTRasym ) has been commonly used to quantify fast exchanging amine CEST signals. However, our results show that MTRasym is greatly affected by the above factors, as well as asymmetric MT and nuclear Overhauser enhancement (NOE) effects. Here, we instead applied a relatively more specific inverse analysis method, named AREX (apparent exchange-dependent relaxation), that has previously been applied only to slow and intermediate exchanging solutes. Numerical simulations and controlled phantom experiments show that, although MTRasym depends on T1w and semi-solid content, AREX acquired in steady state does not, which suggests that AREX is more specific than MTRasym . By combining with a fitting approach instead of using the asymmetric analysis to obtain reference signals, AREX can also avoid contaminations from asymmetric MT and NOE effects. Animal experiments show that these two quantification methods produce differing contrasts between tumors and contralateral normal tissues in rat brain tumor models, suggesting that conventional MTRasym applied in vivo may be influenced by variations in T1w , semi-solid content, or NOE effect. Thus, the use of MTRasym may lead to misinterpretation, while AREX with corrections for competing effects likely enhances the specificity and accuracy of quantification to fast exchanging pools.

To correct the temporal B0 drift in chemical exchange saturation transfer (CEST) imaging in real-time with extra free-induction-decay (FID) readout. The frequency stabilization module of the recently proposed frequency-stabilized CEST (FS-CEST) sequence was further simplified by replacing the original three k-space lines of gradient-echo (GRE) readout with a single k-space line of FID readout. The B0 drift was quantified using the phase difference between the odd and even parts of the FID signal in the frequency stabilization module and then used to update the B0 frequency in the succeeding modules. The proposed FS-CEST sequence with FID readout (FID FS-CEST) was validated in phantoms and 16 human subjects on cross-vendor scanners. In the Siemens experiments, the FID FS-CEST sequence successfully corrected the user-induced B0 drift, generating consistent amide proton transfer-weighted (APTw) images and magnetization transfer ratio asymmetry (MTRasym ) spectra with those from the non-frequency-stabilized CEST (NFS-CEST) sequence without B0 drift. In the Philips experiments, the FID FS-CEST sequence produced more stable APTw images and MTRasym spectra than the NFS-CEST sequence in the presence of practical B0 drift. Quantitatively, the SD of the APTw signal values in the deep gray matter from 15 subjects was 0.26% for the FID FS-CEST sequence compared to 1.03% for the NFS-CEST sequences, with the fluctuations reduced by nearly three-quarters. The proposed FS-CEST sequence with FID readout can effectively correct the temporal B0 drift on cross-vendor scanners.

Introduction. Triple negative breast cancer (TNBC), representing 10-15% of all diagnosed breast cancer (BC) cases, is the most aggressive BC subtype and has higher recurrence rates and therapy resistance. It typically has an inflamed tumor microenvironment and relatively high tumor infiltrating lymphocyte levels. However, T cells are typically less activated compared to non-invasive BC. Hence, we hypothesize that local delivery of immunotherapeutics to activate and reinvigorate T cells can restore the anti-tumor immune response. To this end, we have developed an injectable delivery medium to effectively localize immune agonists to activate, expand, and reinvigorate T cells at the site of the tumor. Methods. The naturally occurring polysaccharide chitosan was crosslinked to form a novel injectable hydrogel (XCSgel). For in vivo tumor treatment, 2.5 × 105 E0771 murine breast cancer cells were implanted subcutaneously on the right flank of C57BL/6 mice. When tumors grew to be 50-100mm3, 5ug and 20ug of interleukin-12 (IL-12) co-formulated in 50uL of XCSgel was delivered intratumorally, with additional treatment groups of 5ug IL-12 in saline, XCSgel alone, and an untreated control. For re-challenge studies, 2.5 × 105 E0771 murine breast cancer cells were implanted subcutaneously on the left flank of mice, with a naïve cohort as a control. For the second tumor study to reinvigorate T cells in mice with larger tumor burdens, 5 × 105 E0771 murine breast cancer cells were implanted subcutaneously on the right flank of C57BL/6 mice. When tumors reach 100-150mm3, 5ug IL-12 in XCSgel, 20ug IL-10 in XCSgel, 5ug IL-12 + 20ug IL-10 in XCSgel, 5ug IL-12 + 20ug IL-10 in saline, and XCSgel will be delivered intratumorally, with an untreated control. Tumor sizes and Kaplan-Meier survival curves were/will be collected across all cohorts. All experiments involving laboratory animals were approved by the Institutional Animal Care and Use Committee at North Carolina State University. Results and Discussion. In the first tumor study, 5ug IL-12 in XCSgel eliminated tumors in 5/5 mice, while 20ug IL-12 in XCSgel eliminated tumors in 4/5 mice and IL-12 in saline eliminated 2/5 tumors. No tumor delay or elimination was observed in untreated and gel alone groups. Upon re-challenge, 5/5 mice in the 5ug IL-12 in XCSgel remained tumor-free over 40 days post-re-challenge, as did 3/4 of the 20ug in XCSgel and 2/2 in IL-12 saline treatment groups. The second intratumoral study is ongoing. Conclusions. A single injection of IL-12 formulated in a novel injectable hydrogel was found to eliminate tumors and stimulate immune memory in a murine triple negative breast cancer model. Results from localized IL-12 and IL-10 cytokine treatment of tumors with larger tumor burdens are ongoing. Given the ability of this localized immunotherapy to induce systemic antitumor immunity, it deserves further consideration as a neoadjuvant treatment prior to breast conserving surgery.Acknowledgements. This work is supported by the NSF Graduate Research Fellowship. Citation Format: Siena M Mantooth, David A Zaharoff. Localized cytokine immunotherapy with a novel injectable hydrogel confers immune memory in murine triple negative breast cancer tumors [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B031.